Water-soluble rhodamine dye conjugates

ABSTRACT

The present invention provides novel, water-soluble, red-emitting fluorescent rhodamine dyes and red-emitting fluorescent energy-transfer dye pairs, as well as labeled conjugates comprising the same and methods for their use. The dyes, energy-transfer dye pairs and labeled conjugates are useful in a variety of aqueous-based applications, particularly in assays involving staining of cells, protein binding, and/or analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.

[0001] This application is a division of pending application Ser. No.09/661,206, filed on Sep. 14, 2000, which is a division of applicationSer. No. 09/433,093, filed on Nov. 3, 1999, now U.S. Pat. No. 6,191,278,issued Feb. 20, 2001, all of which are incorporated herein by reference.

1. FIELD OF THE INVENTION

[0002] The present invention relates generally to fluorescent dyecompounds that are useful as molecular probes. In particular, thepresent invention relates to fluorescent rhodamine dye compounds thatare photostable and highly water-soluble.

2. BACKGROUND OF THE INVENTION

[0003] The non-radioactive detection of nucleic acids utilizingfluorescent labels is an important technology in modem molecularbiology. By eliminating the need for radioactive labels, safety isenhanced and the environmental impact and costs associated with reagentdisposal is greatly reduced. Examples of methods utilizing suchnon-radioactive fluorescent detection include automated DNA sequencing,oligonucleotide hybridization methods, detection ofpolymerase-chain-reaction products, immunoassays, and the like.

[0004] In many applications it is advantageous to employ multiplespectrally distinguishable fluorescent labels in order to achieveindependent detection of a plurality of spatially overlapping analytes,i.e., multiplex fluorescent detection. Examples of methods utilizingmultiplex fluorescent detection include single-tube multiplex DNA probeassays, PCR, single nucleotide polymorphisms and multi-color automatedDNA sequencing. The number of reaction vessels may be reduced therebysimplifying experimental protocols and facilitating the production ofapplication-specific reagent kits. In the case of multi-color automatedDNA sequencing, multiplex fluorescent detection allows for the analysisof multiple nucleotide bases in a single electrophoresis lane therebyincreasing throughput over single-color methods and reducinguncertainties associated with inter-lane electrophoretic mobilityvariations.

[0005] Assembling a set of multiple spectrally distinguishablefluorescent labels useful for multiplex fluorescent detection isproblematic. Multiplex fluorescent detection imposes at least six severeconstraints on the selection of component fluorescent labels,particularly for applications requiring a single excitation lightsource, an electrophoretic separation, and/or treatment with enzymes,e.g., automated DNA sequencing. First, it is difficult to find a set ofstructurally similar dyes whose emission spectra are spectrallyresolved, since the typical emission band half-width for organicfluorescent dyes is about 40-80 nanometers (nm). Second, even if dyeswith non-overlapping emission spectra are identified, the set may stillnot be suitable if the respective fluorescent quantum efficiencies aretoo low. Third, when several fluorescent dyes are used concurrently,simultaneous excitation becomes difficult because the absorption bandsof the dyes are usually widely separated. Fourth, the charge, molecularsize, and conformation of the dyes must not adversely affect theelectrophoretic mobilities of the analyte. Fifth, the fluorescent dyesmust be compatible with the chemistry used to create or manipulate theanalyte, e.g., DNA synthesis solvents and reagents, buffers, polymeraseenzymes, ligase enzymes, and the like. Sixth, the dye must havesufficient photostability to withstand laser excitation.

[0006] Currently available multiple dye sets suitable for use infour-color automated DNA sequencing applications require blue orblue-green laser light to adequately excite fluorescence emissions fromall of the dyes making up the set, e.g., argon-ion lasers. As lower costred lasers become available, a need develops for fluorescent dyecompounds and their nucleic acid conjugates which satisfy the aboveconstraints and are excitable by laser light having a wavelength aboveabout 500 nm.

3. SUMMARY OF THE INVENTION

[0007] These and other objects are furnished by the present invention,which in one aspect provides water-soluble, photostable rhodamine dyecompounds that can be used as labels in a variety of biological andnon-biological assays. Generally, the rhodamine dye compounds of theinvention comprise a rhodamine-type parent xanthene ring substituted atthe xanthene C-9 carbon with a substituted phenyl ring. The substitutedphenyl ring contains three to five substituents including: an orthocarboxyl or sulfonate group; one or more aminopyridinium (“Pyr⁺”)groups; and one alkylthio, arylthio or heteroarylthio group. Thealkylthio, arylthio or heteroarylthio group is believed to be positionedpara to the carboxyl or sulfonate group, with the remaining positionsbeing substituted with Pyr⁺ groups.

[0008] The aminopyridinium groups are attached to the phenyl ring at thepyridinium ring nitrogen and may be substituted or unsubstituted at thepyridinium ring carbons with one or more of a wide variety of the sameor different substituents. The substituents may be virtually any group.However, electron-withdrawing groups (e.g., —NO₂, —F, —Cl, —CN, —CF₃,etc.) should not be attached directly to the pyridinium ring carbons, asthese substituents may adversely affect the synthesis of the rhodaminedyes. Electron-withdrawing groups may be included on a substituent aslong as it is spaced away from the pyridinium ring so as to notadversely affect the synthesis of the dyes. Thus, typical pyridiniumring carbon substituents include, but are not limited to —R, —OR, —SR,—NRR, —S(O)₂O⁻, —S(O)₂OH, −S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X,—C(O)OR, —C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and—C(NR)NRR, where each R is independently hydrogen, (C₁-C₆) alkyl, orheteroalkyl, (C₅-C₁₄) aryl or heteroaryl. The R groups may be furthersubstituted with one or more of the same or different substituents,which are typically selected from the group consisting of —X, —R′, ═O,—OR′, —SR′, ═S, —NRR′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —C(O)R′, —C(O)X, —C(S)R′,—C(S)X, —C(O)OR, —C(O)O—, —C(S)OR′, —C(O)SR, —C(S)SR′, —C(O)NR′R′,—C(S)NR′R and —C(NR)NR′R′, where each X is independently a halogen(preferably —F or —Cl) and each R′ is independently hydrogen, (C₁-C₆)alkyl or heteroalkyl, (C₅-C₁₄) aryl or heteroaryl. Preferably, thepyridium ring carbons are unsubstituted. When substituted, the mostpreferred substituents are the same or different (C₁-C₆) alkyls.

[0009] The amino group of the aminopyridinium groups is located at the4-position of the pyridinium ring. The amino group may be a primary,secondary or tertiary amino group, but is typically a tertiary amino.The nitrogen substituents are typically (C₁-C₆) alkyl groups orheteroalkyl groups, and may be the same or different. Alternatively, thenitrogen is substituted with an alkyldiyl or heteroalkyldiyl bridgehaving from 2 to 5 backbone atoms such that the substituents and thenitrogen atom taken together form a ring structure, which may besaturated or unsaturated, but is preferably saturated. The bridgesubstituent may be branched or straight-chain, but is preferablystraight-chain, e.g., ethano, propano, butano, etc. The ring structuremay contain, in addition to the nitrogen atom of the aminopyridinium,one or more heteroatoms, which are typically selected from the groupconsisting of O, S and N. When the nitrogen atom is not included in aring structure, the amino group is preferably dimethylamino. When thenitrogen atom is included in a ring structure, the ring is preferably amorpholino or piperazine ring. Particularly preferred Pyr⁺ groups are4-(dimethylamino)pyridinium, 4-(morpholino) pyridinium, and1-methyl-4-piperazinylpyridinium.

[0010] The alkylthio, arylthio or heteroarylthio group is attached tothe phenyl ring via the sulfur atom and may also be substituted with oneor more of the same or different substituents. The nature of thesubstituents will depend upon whether the group is an alkylthio,arylthio or heteroarylthio. The alkyl chain of an alkylthio group may besubstituted with virtually any substituent, including, but not limitedto, —X, —R, =O, —OR, —SR, ═S, —NRR, ═NR, —CX₃, —CN, —OCN, —SCN, —NCO,—NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)2R, —C(O)R, —C(O)X,—C(S)R, —C(S)X, —C(O)OR, —C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR,—C(S)NRR and —C(NR)NRR, where each X is independently a halogen(preferably —F or —Cl) and each R is independently hydrogen, (C₁-C₆)alkyl or heteroalkyl, (C₅-C₁₄) aryl or heteroaryl. The R groups may befurther substituted with one or more of the same or differentsubstituents, which are typically selected from the group consisting of—X, —R′, ═O, —OR, —SR′, ═S, —NRR, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO,—NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —C(O)R′,—C(O)X, —C(S)R′, —C(S)X, —C(O)OR′, —C(O)O⁻, —C(S)OR′, —C(O)SR′,—C(S)SR′, —C(O)NR′R, —C(S)NR′R′ and —C(NR)NR′R′, where each X isindependently a halogen (preferably —F or —Cl) and each R′ isindependently hydrogen, (C₁-C₆) alkyl or heteroalkyl, (C₅-C₁₄) aryl orheteroaryl. Due to synthetic constraints, when the group is an arylthioor heteroalylthio, the aryl or heteroaryl rings should not be directlysubstituted with halogens, although halogens may be included in thesubstituent (e.g., a haloalkyl). Thus, when the group is an arylthio ora heteroarylthio, typical substituents include any of the above-listedalkylthio substituents, with the exception of halogen.

[0011] The rhodamine dyes of the invention may include a linker L thatcan be used to conjugate the dyes, preferably by way of covalentattachment, to other compounds or substances, such as peptides,proteins, antibodies, nucleoside/tides, polynucleotides, polymers,particles, etc. The identity of linker L will depend upon the nature ofthe desired conjugation. For example, the conjugation may be: (i)mediated by ionic interactions, in which case linker L is a chargedgroup; (ii) mediated by hydrophobic interactions, in which case L is ahydrophobic moiety; (iii) mediated by covalent attachment, in which caseL is a reactive functional group (R_(X) ) that is either capable offorming a covalent linkage with another complementary functional group(F_(x) ) or is capable of being activated so as to form a covalentlinkage with complementary functional group F_(x) ; or (iv) mediatedthrough the use of pairs of specific binding molecules, such as biotinand avidinistreptavidin, in which case linker L is one member of thepair, e.g., biotin. Linker L is attached to the rhodamine dyes of theinvention at the rhodamine-type parent xanthene ring and/or it isincluded as a substituent on the alkylthio, arylthio or heteroarylthiogroup substituting the fully substituted phenyl ring. When linker L isattached to the rhodamine-type parent xanthene ring, it is typicallyattached to a xanthene nitrogen or at the xanthene C4 carbon. Therhodamine dyes may have multiple linking moieties, but preferably haveonly a single linking moiety.

[0012] Depending upon the particular application, linker L may beattached directly to the rhodamine dye, or indirectly through one ormore intervening atoms that serve as a spacer. Linker L can behydrophobic or hydrophilic, long or short, rigid, semirigid or flexible,depending upon the particular application. When L is positioned at thealkylthio, arylthio or heteroarylthio group, it is preferably attacheddirectly to the molecule. In this latter embodiment, L is a bond.

[0013] The new, fully substituted phenyl rings described herein can beused to replace the “bottom ring” or “bottom substituent,” ie., thesubstituent attached to the xanthene C9 carbon, of virtually anyrhodamine dye that is known in the art or that will be later developed.Thus, the new, fully substituted phenyl rings described herein can becovalently attached to the C-9 position of virtually any rhodamine-typeparent xanthene ring that is now known or that will be later developedto yield a rhodamine dye without longer absorption and emission maximaand with greater water-solubility. As the new bottom rings do notdeleteriously affect the photostability properties that arecharacteristic of rhodamine dyes, the new dyes are also highlyphotostable. Exemplary rhodamine-type parent xanthene rings that cancomprise the rhodamine dyes of the invention include, by way of exampleand not limitation, the xanthene rings (“top rings”) of the rhodaminedyes described in U.S. Pat. No. 5,936,087; U.S. Pat. No. 5,750,409; U.S.Pat. No. 5,366,860; U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,840,999;U.S. Pat. No. 5,847,162; U.S. application Ser. No. 09/038,191, filedMar. 10, 1998; U.S. application Ser. No. 09/277,793, filed Mar. 27,1999; U.S. application Ser. No. 09/325,243, filed Jun. 3, 1999; PCTPublication WO 97/36960; PCT Publication WO 99/27020; Sauer et al.,1995, J. Fluorescence 5(3):247-261; Arden-Jacob, 1993, Neue LanwelligeXanthen-Farbstoffe für Fluoreszenzsonden und Farbstoff Laser, VerlagShaker, Germany, and Lee et al., 1992, Nucl. Acids Res.20(10):2471-2483. Preferred rhodamine-type parent xanthene rings arefluorescent.

[0014] In another aspect, the invention provides labeled conjugatescomprising a rhodamine dye according to the invention and anothermolecule or substance. The rhodamine dye is conjugated to the othermolecule or substance, typically via covalent attachment, through linkerL, as previously described. Once conjugated, the rhodamine dye providesa convenient fluorescent label for subsequent detection. The rhodaminedyes of the invention can be used to fluorescently label a wide varietyof molecules and substances, including but not limited to, amino acids,peptides, proteins, antibodies, enzymes, receptors, nucleosides/tides,nucleoside/tide analogs, polynucleotides, polynucleotide analogs,nucleic acids, carbohydrates, lipids, steroids, hormones, vitamins,drugs, metabolites, toxins, organic polymers, etc. The dyes can also beused to label particles such as solid phase synthesis substrates,nanoparticles, microspheres or liposomes. In embodiments involvingnanoparticles, microspheres and/or liposomes, the due need not include alinking moiety. It can be incorporated into the various particles duringtheir formation. The molecule or substance may be labeled with one ormore rhodamine dyes of the invention, which may be the same ordifferent.

[0015] In one embodiment, a rhodamine dye of the invention is covalentlyconjugated to another dye compound to form an energy-transfer dye pair.The energy-transfer dye pair can be further conjugated to othermolecules or substances, as described above, to provide anenergy-transfer label. The energy-transfer dye pair generally comprisesa donor dye (DD), an acceptor dye (AD), and a linkage linking the donorand acceptor dyes. The donor dye is capable of absorbing light at afirst wavelength and emitting excitation energy in response. Theacceptor dye is capable of absorbing the excitation energy emitted bythe donor dye and fluorescing at a second wavelength in responsethereto. While in many instances the emission wavelength of the donordye and the excitation wavelength of the acceptor dye will overlap, suchoverlap is not required. The acceptor dye need only fluoresce inresponse to the donor dye absorbing light, regardless of the mechanismof action. The linkage serves to facilitate efficient energy transferbetween the donor and acceptor dyes. According to this aspect of theinvention, at least one of the donor and acceptor dyes is a rhodaminedye according to the invention. Preferably, the acceptor dye is arhodamine dye according to the invention and the donor dye is a xanthenedye, most typically a fluorescein dye. The exact nature or identity ofthe donor dye will depend upon the excitation and emission properties ofthe rhodamine acceptor dye, and will be apparent to those having skillin the art. When covalently conjugated to enzymatically-incorporatablenucleotides, such as dideoxynucleotide 5′-triphosphates, i.e.“terminators”, such energy-transfer dyes are ideally suited for use insequencing nucleic acids.

[0016] Since the rhodamine dyes of the invention may comprise virtuallyany rhodamine-type parent xanthene ring, the dyes cover a broad range ofthe visible spectrum, ranging from green to red. Thus, both dyes of anenergy-transfer dye pair may be rhodamine dyes of the invention. In thisembodiment, one dye of the invention acts as the donor and another asthe acceptor, depending upon their spectral properties.

[0017] In another embodiment, a rhodamine dye of the invention, or anenergy-transfer dye pair including a rhodamine dye of the invention, iscovalently conjugated to a nucleoside/tide, nucleoside/tide analog,polynucleotide or polynucleotide analog to form a labeled conjugatetherewith. The dye or dye pair is typically conjugated to the nucleobasemoiety of the respective nucleoside/tide, polynucleotide or analog, butmay be conjugated to other portions of the molecule, such as the5′-terminus, 3′-terminus and/or the phosphate ester internucleosidelinkage.

[0018] In one preferred embodiment, the labeled conjugate is a labeledpolynucleotide or polynucleotide analog that can be used as a primer forgenerating labeled primer extensions products via template-directedenzymatic synthesis reactions. In another preferred embodiment, thelabeled conjugate is a labeled terminator. When used in conjunction withenzymatically-extendable nucleotides or nucleotide or analogs,appropriate polymerizing enzymes and a primed template nucleic acid,such labeled terminators can be used to generate a series of labeledprimer extension products via template-directed enzymatic synthesis forapplications such as nucleic acid sequencing.

[0019] In a final aspect, the invention provides methods of using therhodamine dyes or energy-transfer dye pairs of the invention to sequencea target nucleic acid. The method generally comprises forming a seriesof differently-sized primer extension products labeled with a rhodaminedye or energy-transfer dye pair of the invention, separating the seriesof differently-sized labeled extension products, typically based onsize, and detecting the separated labeled extension products based onthe fluorescence of the label. The sequence of the target nucleic acidis then assembled according to known techniques.

[0020] The series of differently-sized labeled extension products can beconveniently generated by enzymatically extending a primer-target hybridaccording to well-known methods. For example, the series of labeledextension products can be obtained using a primer labeled with arhodamine dye or dye pair of the invention and enzymatically extendingthe labeled primer-target hybrid in the presence of a polymerase, amixture of enzymatically-extendable nucleotides or nucleotide analogscapable of supporting continuous primer extension (e.g., dATP, dGTP,dCTP and dUTP or dTTP) and at least one, typically unlabeled, terminatorthat terminates primer extension upon incorporation (e.g., a ddNTP).Alternatively, the series of labeled extension products can be obtainedusing an unlabeled primer and enzymatically extending the unlabeledprimer-target hybrid in the presence of a polymerase, a mixture ofenzymatically-extendable nucleotides or nucleotide analogs capable ofsupporting continuous primer extension and at least one terminatorlabeled with a rhodamine dye or energy-transfer dye pair of theinvention. In either embodiment, the polymerase serves to extend theprimer with enzymatically-extendable nucleotides or nucleotide analogsuntil a terminator is incorporated, which terminates the extensionreaction. Once terminated, the series of labeled extension products areseparated, typically based on size, and the separated labeled extensionproducts detected based on the fluorescence of the labels.

[0021] In a particularly advantageous embodiment of this method, amixture of four different terminators are used in a single extensionreaction. Each different terminator is capable of terminating primerextension at a different template nucleotide, e.g., a mixture of7-deaza-ddATP, ddCTP, 7-deaza-ddGTP and ddTTP or ddUTP, and is labeledwith a different, spectrally-resolvable fluorophore, at least one ofwhich is a rhodamine dye or energy-transfer dye pair according to theinvention. According to this embodiment, an unlabeled primer-targetnucleic acid hybrid is enzyrnmatically extended in the presence of apolymerase, a mixture of enzymnatically-extendable nucleotides ornucleotide analogs capable of supporting continuous primer extension anda mixture of the four different labeled terminators. Followingseparation based on size, a series of separated labeled extensionproducts is obtained in which the emission properties (i.e., color) ofeach separated extension product reveals the identity of its 3v-terminalnucleotide. In a particularly preferred embodiment, all of the labeledterminators are excitable using a single light source.

[0022] Alternatively, terminators may be used in the absence ofenzymatically-extendable nucleotides. In this instance, the primer isextended by only a single base. Again, the primer may be labeled, or,alternatively, one or more of the terminators may be labeled.Preferably, a mixture of four different labeled terminators is used, asdescribed above. These “mini sequencing” embodiments are particularlyuseful for identifying polymorphisms in chromosomal DNA or cDNA.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 provides single color sequencing data obtained with plasmidpGEM, an unlabeled sequencing primer and the labeled terminator6-FAM-196-7-deaza-ddATP on an ABI PRISM Model 310 sequencer (PEBiosystems, Foster City, Calif.).

[0024]FIG. 2 provides four-color sequencing data obtained with plasmidpGEM1, an unlabeled sequencing primer and a mixture of four, spectrallyresolvable, 3′-fluoro, labeled terminators: 6-FAM-196-7-deaza-ddATP;5-FAM-dR110-7-deaza-ddGTP; 6-FAM-dJON-ddTTP; and 6-FAM-dROX-ddCTP on anABI PRISM Model 310 sequencer (PE Biosystems, Foster City, Calif.).

[0025]FIG. 3 provides the fluorescence emission spectra (H₂O) of fourdyes with emission maxima from left to right: 236 (Emax=545 nm), 190(Emax=650 nm), 196 (Emax=661 nm), 232 (Emax=665 nm). The ordinate axisis fluorescence units and the abscissa axis is emission wavelength innm.

[0026]FIG. 4 is a plot of a standard curve of the log v. log graph ofthe average fluorescence intensity in detection at FL1 (650-685 nm) vs.pg/ml of the IL-8 peptide by a fluorescence-linked immunosorbent assay.

5. DETAILED DESCRIPTION OF THE INVENTION

[0027] 5.1 Abbreviations

[0028] The abbreviations used throughout the specification to refer tocertain nucleobases, nucleosides and/or nucleotides are those commonlyemployed in the art and are as indicated below: Expression Abbreviationadenine A 7-deazaadenine 7-deaza-A N⁶-Δ²-isopentenyladenine 6iAN⁶-Δ²-isopentenyl-2-methylthioadenine 2ms6iA cytosine C guanine G6-thioguanine 6sG 7-deazaguanine 7-deaza-G N²-dimethylguanine 2dmG7-methylguanine 7mG thymine T 4-thiothymine 4sT uracil U dihydrouracil D4-thiouracil 4sU base Y Y ribonucleoside-5′-triphosphate NTPadenosine-5′-triphosphate ATP 7-deazaadenosine-5′-triphosphate7-deaza-ATP cytidine-5′-triphosphate CTP guanosine-5′-triphosphate GTP7-deazaguanosine-5′-triphosphate 7-deaza-GTP thymidine-5′-triphosphateTTP uridine-5′-triphosphate UTP 2′-deoxyribonucleoside-5′-triphosphatedNTP 2′-deoxyadenosine-5′-triphosphate dATP2′-deoxy-7-deazaadenosine-5′-triphosphate 7-deaza-dATP2′-deoxycytidine-5′-triphosphate dCTP 2′-deoxyguanosine-5′-triphosphatedGTP 2′-deoxy-7-deazaguanosine-5′-triphosphate 7-deaza-dGTP2′-deoxythymidine-5′-triphosphate dTTP 2′-deoxyuridine-5′-triphosphatedUTP 2′,3′-dideoxyribonucleoside-5′-triphosphate ddNTP2′,3′-dideoxyadenosine-5′-triphosphate ddATP2′,3′-dideoxy-7-deazaadenosine-5′-triphosphate 7-deaza-ddATP2′,3′-dideoxycytidine-5′-triphosphate ddCTP2′,3′-dideoxyguanosine-5′-triphosphate ddGTP2′,3′-dideoxy-7-deazaguanosine-5′-triphosphate 7-deaza-ddGTP2′,3′-dideoxythymidine-5′-triphosphate ddTTP2′,3′-dideoxyuridine-5′-triphosphate ddUTP

[0029] 5.2 Definitions

[0030] In general, the terms used herein to describe the presentinvention rely on definitions as understood and used by those skilled inthe art. In particular, chemical structures and substructures aredescribed according to IUPAC recommendations (“Nomenclature of OrganicCompounds: A Guide to IUPAC Recommendations 1993, R. Panico, W. H.Powell, and Jean-Claude Richer, Eds., Blackwell Science, Ltd., Oxford,U.K.). As used herein, the following terms are intended to have thefollowing meanings:

[0031] “Spectrally Resolvable:” means, in reference to a set offluorescent dyes and/or energy-transfer dye pairs (collectively referredto herein as “dyes” or “labels”), that the fluorescence emission bandsof the respective dyes are sufficiently distinct, ie., sufficientlynon-overlapping, that the dyes, either alone or when conjugated to othermolecules or substances, are distinguishable from one another on thebasis of their fluorescence signals using standard photodetectionsystems such as photodetectors employing a series of band pass filtersand photomultiplier tubes, charged-coupled devices (CCD), spectrographs,etc., as exemplified by the systems described in U.S. Pat. Nos.4,230,558 and 4,811,218 or in Wheeless et al., 1985, Flow Cytometry:Instrumentation and Data Analysis, pp. 21-76, Academic Press, New York.Preferably, all of the dyes comprising a spectrally resolvable set ofdyes are excitable by a single light source.

[0032] “Nucleobase:” refers to a substituted or unsubstitutednitrogen-containing parent heteroaromatic ring of a type that iscommonly found in nucleic acids. Typically, but not necessarily, thenucleobase is capable of forming Watson-Crick and/or Hoogsteen hydrogenbonds with an appropriately complementary nucleobase. Exemplarynucleobases include, but are not limited to, purines such as2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine,N⁶-Δ²-isopentenyladenine (6iA), N⁶-Δ²-isopentenyl-2-methylthioadenine(2ms6iA), N⁶-methyladenine, guanine (G), isoguanine, N²-dimethylguanine(dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG),hypoxanthine and O⁶-methylguanine; 7-deaza-purines such as7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidinessuch as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),4-thiothymine (4sT), 5,6-dihydrothymine, O⁴-methylthymine, uracil (U),4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indolessuch as nitroindole and 4-methylindole; pyrroles such as nitropyrrole;nebularine; base (Y); etc. Additional exemplary nucleobases can be foundin Fasman, 1989, Practical Handbook of Biochemistry and MolecularBiology, pp. 385-394, CRC Press, Boca Raton, Fla., and the referencescited therein. Preferred nucleobases are purines, 7-deazapurines andpyrimidines. Particularly preferred nucleobases are the normalnucleobases, defined infra, and their common analogs, e.g., 2ms6iA, 6iA,7-deaza-A, D, 2dmG, 7-deaza-G, 7mG, hypoxanthine, 4sT, 4sU and Y.

[0033] “Normal Nucleobase:” refers to a nucleobase that isnaturally-occurring and encoding, i.e., adenine, cytosine, guanine,thymine or uracil.

[0034] “Nucleoside:” refers to a compound consisting of a nucleobasecovalently linked, typically via a heteroaromatic ring nitrogen, to theCl′ carbon of a pentose sugar. Typical pentose sugars include, but arenot limited to, those pentoses in which one or more of the carbon atomsare each independently substituted with one or more of the same ordifferent —R, —R, —NRR or halogen groups, where each R is independentlyhydrogen, (C₁-C₆) alkyl or (C₅-C₁₄) aryl. The pentose sugar may besaturated or unsaturated. Exemplary pentose sugars include, but are notlimited to, ribose, 2′-deoxyribose, 2′-(C₁-C₆)alkylribose,2′-(C₁-C₆)alkoxyribose, 2′-(C₅-C₁₄)aryloxyribose, 2′,3′-dideoxyribose,2′,3′-didehydroribose, 2′-deoxy-3′-haloribose, 2′-deoxy-3′-fluororibose,2′-deoxy-3′-chlororibose, 2′-deoxy-3′-aminoribose,2′-deoxy-3′-(C₁-C₆)alkylribose, 2′-deoxy-3′-(C₁-C₆)alkoxyribose and2′-deoxy-3′-(C₅-C₁₄)aryloxyribose.

[0035] When the nucleobase is a purine or a 7-deazapurine, the pentosesugar is attached to the N9-position of the nucleobase. When thenucleobase is a pyrimidine, the pentose sugar is attached to theNi-position of the nucleobase (see, e.g., Komberg and Baker, 1992, DNAReplication, 2^(nd) Ed., Freeman, San Francisco), except forpseudouridine, in which the pentose sugar is attached to the C5 positionof the uracil nucleobase. Preferred nucleosides are those in which thenucleobase is a purine, a 7-deazapurine, a pynmidine, a normalnucleobase or a common analog thereof and the pentose sugar is one ofthe exemplary pentose sugars listed above.

[0036] “Normal Nucleoside:” refers to a compound consisting of a normalnucleobase covalently linked via the Ni (C, T or U) or N9 (A or G)position of the nucleobase to the Cl′ carbon of ribose or2′-deoxyribose.

[0037] “Nucleoside Analog:” refers to a nucleoside in which the pentosesugar is replaced with a pentose sugar analog. Exemplary pentose sugaranalogs include, but are not limited to, substituted or unsubstitutedfuranoses having more or fewer than 5 ring atoms, e.g. erythroses andhexoses, and substituted or unsubstituted 3-6 carbon acyclic sugars. Oneor more of the carbon atoms may be independently substituted with one ormore of the same or different —R, —R, —NRR or halogen groups, where eachR is independently hydrogen, (C₁-C₆) alkyl or (C₅-C₁₄) aryl.

[0038] “Nucleotide:” refers to a nucleoside in which one or more,typically one, of the pentose carbons is substituted with a phosphateester having the formula:

[0039] where a is an integer from 0 to 4. Preferably, a is 2 and thephosphate ester is attached to the 3′- or 5′-carbon of the pentose.Particularly preferred nucleotides are those which are(atically-extendable or enzymatically incorporatable (defined infra).

[0040] “Normal Nucleofide:” refers to a normal nucleoside in which the3′- or 5′-carbon of the ribose or 2′-deoxyribose sugar is substitutedwith a phosphate ester having the formula:

[0041] where a is an integer from 0 to 2. Preferred normal nucleotidesare those in which a is 2 and the phosphate ester is attached to the5′-carbon of the ribose (an NTP) or 2′-deoxyribose (a DNTP).

[0042] “Nucleotide Analog:” refers to a nucleotide in which the pentosesugar and/or one or more of the phosphate esters is replaced with itsrespective analog. Exemplary pentose sugar analogs are those previouslydescribed in conjunction with nucleoside analogs. Exemplary phosphateester analogs include, but are not limited to, alkylphosphonates,methylphosphonates, phosphoramidates, phosphotriesters,phosphorothioates, phosphorodithioates, phosphoroselenoates,phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates,phosphoroamidates, boronophosphates, etc., including any associatedcounterions, if present.

[0043] Also included within the definition of “nucleotide analog” arenucleobase-containing molecules which can be polymerized intopolynucleotide analogs in which the DNA/RNA phosphate ester and/or sugarphosphate ester backbone is replaced with a different type of linkage.Such polynucleotide analogs are described in more detail, infra.

[0044] “Enzymatically-Incorporatable Nucleotide or Nucleotide Analog:”refers to a nucleotide or nucleotide analog which is capable of actingas a substrate for a polymerizing enzyme in a template-directed nucleicacid synthesis reaction such that it is incorporated by the enzyme intoa nascent polynucleotide or polynucleotide analog chain. Typicalenzymatically-incorporatable nucleotides and nucleotide analogs arethose in which the sugar is a pentose. Preferredenzymatically-incorporatable nucleotides are those in which thenucleobase is a purine, a 7-deazapurine, a pyrimidine, a normalnucleobase or a common analog thereof and the pentose sugar is apentose-5′-triphosphate, such as NTPs, dNTPs and ddNTPs.

[0045] “Enzymatically-Extendable Nucleotide or Nucleotide Analog:”refers to an enzymatically-incorporatable nucleotide or nucleotideanalog that, once incorporated into the nascent polynucleotide orpolynucleotide analog chain, supports incorporation of furthernucleotides or nucleotide analogs. Thus, enzymatically-extendablenucleotides or nucleotide analogs have a hydroxyl group that is capableof forming a covalent linkage with another, subsequent nucleotide ornucleotide analog. Typical enzymatically-extendable nucleotides andnucleotide analogs are those in which the sugar is a pentose. Preferredenzymatically-extendable nucleotides are those in which the nucleobaseis a purine, a 7-deazapurine, a pyrimidine, a normal nucleobase or acommon analog thereof and the pentose sugar is a3′-hydroxypentose-5′-triphosphate, such as NTPs and dNTPs.

[0046] A mixture of enzymatically-extendable nucleotides or nucleotideanalogs is said to support continuous primer extension when the mixturecontains an enzymatically-extendable nucleotide or nucleotide analogcomplementary to each base of the template polynucleotide, e.g., amixture of dATP, dGTP, dCTP and dUTP or dTTP.

[0047] “Terminator:” refers to an enzymatically-incorporatablenucleotide or nucleotide analog that, once incorporated into the nascentpolynucleotide chain, terminates further chain extension. Typicalterminators are those in which the nucleobase is a purine, a7-deaza-purine, a pyrimidine, a normal nucleobase or a common analogthereof and the sugar moiety is a pentose which includes a3′-substituent that blocks further synthesis, such as a ddNTP.Substituents that block further synthesis include, but are not limitedto, amino, deoxy, halogen, alkoxy and aryloxy groups. Exemplaryterminators include, but are not limited to, those in which thesugar-phosphate ester moiety is 3′-(C₁-C₆)alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkylribose-5′-triphosphate,2-deoxy-3′-(C₁-C₆)alkoxyribose-5-triphosphate,2′-deoxy-3′-(C₁-C₁₄)aryloxyribose-5′-triphosphate,2′-deoxy-3′-halofibose-5′-triphosphate,2′-deoxy-3′-aminoribose-5′-triphosphate,2′,3′-dideoxyribose-5′-triphosphate or2′,3′-didehydroribose-5′-triphosphate.

[0048] “Nucleoside/tide:” refers to a nucleoside and/or a nucleotideand/or a mixture thereof.

[0049] “Polynucleotide:” refers to a linear polymeric chain ofnucleoside monomer units that are covalently connected to one another byphosphate ester intemucleoside linkages. Unless stated otherwise,“polynucleotide” as used herein includes polymers of any length,including oligonucleotides, polynucleotides and nucleic acids as thoseterms are commonly used in the art. Where polynucleotides of specificsize ranges are intended, the number of monomer units is specificallydelineated. Thus, polynucleotides according to the invention can rangein size from a few monomer units (e.g., 4 to 40), to several hundreds ofmonomer units, to several thousands of monomer units, or even moremonomer units. Whenever a polynucleotide is represented by a sequence ofletters, e.g. “ATGCCTG,” it will be understood that the sequence ispresented in the 5′→3′ direction. 2′-Deoxyribopolynucleotides arepreceded with the letter “d,” e.g. “d(ATGCCTG).”

[0050] Polynucleotides may be comprised of a single type of sugarmoiety, as in the case of RNA and DNA, or mixtures of different sugarmoieties, as in the case of RNA/DNA chimeras. Preferred olynucleotidesare ribopolynucleotides and 2′-deoxyribopolynucleotides according to thestructural formulae below:

[0051] wherein:

[0052] each B is independently a nucleobase, preferably a purine, a7-deazapurine, a pyrimidine, a normal nucleobase or a common analogthereof,

[0053] each m defines the length of the respective polynucleotide andcan range from zero to thousands, to tens of thousands, or even more;

[0054] each R is independently selected from the group consisting ofhydrogen, halogen, —R″, —OR″, and —NR″R″, where each R″ is independently(C₁-C₆) alkyl or (C₁-C₁₄) aryl, or two adjacent Rs are taken together toform a bond such that the ribose sugar is 2′,3′-didehydroribose; and

[0055] each R′ is independently hydroxyl or

[0056] , where a is zero, one or two.

[0057] In the preferred ribopolynucleotides and2′-deoxyribopolynucleotides illustrated above, the nucleobases B arecovalently attached to the Cl′ carbon of the sugar moiety as previouslydescribed.

[0058] “Polynucleotide Analog:” refers to a polynucleotide in which atleast one nucleoside monomer unit is a nucleoside analog and/or at leastone phosphate ester intemucleoside linkage is a phosphate ester analog,as previously defined. Also included within the definition ofpolynucleotide analogs are polymers in which the phosphate ester and/orsugar phosphate ester intemucleoside linkages are replaced with othertypes of linkages, such as N-(2-aminoethyl)-glycine amides and otheramides (see, e.g., Nielsen et al., 1991, Science 254:1497-1500; WO92/20702; U.S. Pat. No. 5,719,262; U.S. Pat. No. 5,698,685; morpholinos(see U.S. Pat. No. 5,698,685; U.S. Pat. No. 5,378,841; U.S. Pat. No.5,185,144); carbamates (see Stirchak & Summerton, 1987, J. Org. Chem.52:4202); methylene(methylimino) (see Vasseur et al., 1992, J. Am. Chem.Soc. 114:4006); 3′-thioformacetals (see Jones et al., 1993, J. Org.Chem. 58:2983); sulfamates (see U.S. Pat. No. 5,470,967); and others(see, e.g., U.S. Pat. No. 5,817,781; Frier & Altman, 1997, Nucl. AcidsRes. 25:4429 and the references cited therein).

[0059] “Alkyl:” refers to a saturated or unsaturated, branched,straight-chain or cyclic monovalent hydrocarbon radical derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane, alkene or alkyne. Typical alkyl groups include, but are notlimited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propylssuch as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl,prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls suchas butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl,cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“aLkenyl” and/or “alkynyl” is used, as defined below. In preferredembodiments, the alkyl groups are (C₁-C₆) alkyl.

[0060] “Alkanyl:” refers to a saturated branched, straight-chain orcyclic alkyl radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Typical alkanyl groups include,but are not limited to, methanyl; ethanyl; propanyls such aspropan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butyanylssuch as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl(isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; andthe like. in preferred embodiments, the alkanyl groups are (C₁-C₆)alkanyl.

[0061] “Akenyl:” refers to an unsaturated branched, straight-chain orcyclic alkyl radical having at least one carbon-carbon double bondderived by the removal of one hydrogen atom from a single carbon atom ofa parent alkene. The radical may be in either the cis or transconformation about the double bond(s). Typical alkenyl groups include,but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl ,prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like. Inpreferred embodiments, the alkenyl group is (C₂-C₆) alkenyl.

[0062] “Alkynyl:” refers to an unsaturated branched, straight-chain orcyclic alkyl radical having at least one carbon-carbon triple bondderived by the removal of one hydrogen atom from a single carbon atom ofa parent alkyne. Typical alkynyl groups include, but are not limited to,ethynyl; propynyls such as prop-1-yn-1-yl , prop-2-yn-1-yl, etc.;butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; andthe like. In preferred embodiments, the alkynyl group is (C₂-C₆)alkynyl.

[0063] “Alkyldiyl:” refers to a saturated or unsaturated, branched,straight-chain or cyclic divalent hydrocarbon radical derived by theremoval of one hydrogen atom from each of two different carbon atoms ofa parent alkane, alkene or alkyne, or by the removal of two hydrogenatoms from a single carbon atom of a parent alkane, alkene or alkyne.The two monovalent radical centers or each valency of the divalentradical center can form bonds with the same or different atoms. Typicalalkyldiyls include, but are not limited to methandiyl; ethyldiyls suchas ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Inpreferred embodiments, the alkyldiyl group is (C₁-C₆) alkyldiyl. Alsopreferred are saturated acyclic alkanyldiyl radicals in which theradical centers are at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl(butano); and the like (also referred to as alkylenos, defined infra).

[0064] “Alkyleno:” refers to a straight-chain alkyldiyl radical havingtwo terminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. Typical alkyleno groupsinclude, but are not limited to, methano; ethylenos such as ethano,etheno, ethyno; propylenos such as propano, prop[1]eno, propa[1,2]dieno,prop[1]yno, etc.; butylenos such as butano, but[1]eno, but[2]eno,buta[1,3]dieno, but[1]yno, but[2]yno, but[1,3]diyno, etc.; and the like.Where specific levels of saturation are intended, the nomenclaturealkano, alkeno and/or alkyno is used. In preferred embodiments, thealkyleno group is (C₁-C₆) alkyleno.

[0065] “Heteroalkvl Heteroalkanyl Heteroalkenyl. Heteroalkanyl,Heteroalkyldiyl and Heteroalkyleno:” refer to alkyl, alkanyl, alkenyl,alkynyl, alkyldiyl and alkyleno radicals, respectively, in which one ormore of the carbon atoms are each independently replaced with the sameor different heteroatomic groups. Typical heteroatomic groups which canbe included in these radicals include, but are not limited to, —O—, —S—,—O—O—, —S—S—, —O—S—, —NR′—, ═N—N′, —N═N—, —N(O)N—, —N═N—NR′—, —PH—,—P(O)₂—, —O—P(O)₂—, —SH₂—, —S(O)₂—, —SnH₂— an the like, where each R′ isindependently hydrogen, alkyl, alkanyl, alkenyl, alkynyl, aryl,arylaryl, arylalkyl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl as defined herein.

[0066] “Acyclic Heteroatomic Bridge:” refers to a divalent bridge inwhich the backbone atoms are exclusively heteroatoms. Typical acyclicheteroatomic bridges include, but are not limited to, any of the variousheteroatomic groups listed above, either alone or in combinations.

[0067] “Parent Aromatic Ring System:” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated T electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, indane, indene, phenalene, etc. Typical parent aromaticring systems include, but are not limited to, aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexalene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

[0068] “Aryl:” refers to a monovalent aromatic hydrocarbon radicalderived by the removal of one hydrogen atom from a single carbon atom ofa parent aromatic ring system. Typical aryl groups include, but are notlimited to, radicals derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In preferredembodiments, the aryl group is (C₅-C₁₄) aryl, with (C₅-C₁₀) being evenmore preferred. Particularly preferred aryls are phenyl and naphthyl.

[0069] “Aryldiyl:” refers to a divalent aromatic hydrocarbon radicalderived by the removal of one hydrogen atom from each of two differentcarbon atoms of a parent aromatic ring system or by the removal of twohydrogen atoms from a single carbon atom of a parent aromatic ringsystem. The two monovalent radical centers or each valency of thedivalent center can form bonds with the same or different atom(s).Typical aryldiyl groups include, but are not limited to, divalentradicals derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene, and the like. In preferred embodiments,the aryldiyl group is (C₅-C₁₄) aryldiyl, with (C₅-C₁₀) being even morepreferred. The most preferred aryldiyl groups are divalent radicalsderived from benzene and naphthalene, especially phena-1,4-diyl,naphtha-2,6-diyl and naphtha-2,7-diyl.

[0070] “Aryleno:” refers to a divalent bridge radical having twoadjacent monovalent radical centers derived by the removal of onehydrogen atom from each of two adjacent carbon atoms of a parentaromatic ring system. Attaching an aryleno bridge radical, e.g. benzeno,to a parent aromatic ring system, e.g. benzene, results in a fusedaromatic ring system, e.g naphthalene. The bridge is assumed to have themaximum number of non-cumulative double bonds consistent with itsattachment to the resultant fused ring system. In order to avoiddouble-counting carbon atoms, when an aryleno substituent is formed bytaking together two adjacent substituents on a structure that includesalternative substituents, the carbon atoms of the aryleno bridge replacethe bridging carbon atoms of the structure. As an example, consider thefollowing structure:

[0071] wherein:

[0072] R′, when taken alone is hydrogen, or when taken together with R²is (C₅-C₁₄) aryleno; and

[0073] R², when taken alone is hydrogen, or when taken together with R′is (C₅-C₁₄) aryleno.

[0074] When R′ and R² are each hydrogen, the resultant compound isbenzene. When R′ taken together with R² is C₆ aryleno (benzeno), theresultant compound is naphthalene. When R′ taken together with R² is C₁₀aryleno (naphthaleno), the resultant compound is anthracene orphenanthrene. Typical aryleno groups include, but are not limited to,aceanthryleno, acenaphthyleno, acephenanthryleno, anthraceno, azuleno,benzeno (benzo), chryseno, coroneno, fluorantheno, fluoreno, hexaceno,hexapheno, hexaleno, as-indaceno, s-indaceno, indeno, naphthaleno(naphtho), octaceno, octapheno, octaleno, ovaleno, penta-2,4-dieno,pentaceno, pentaleno, pentapheno, peryleno, phenaleno, phenanthreno,piceno, pleiadeno, pyreno, pyranthreno, rubiceno, triphenyleno,trinaphthaleno, and the like. Where a specific connectivity is intended,the involved bridging carbon atoms (of the aryleno bridge) are denotedin brackets, e.g., [1,2]benzeno ([1,2]benzo), [1,2]naphthaleno,[2,3]naphthaleno, etc. Thus, in the above example, when R′ takentogether with R² is [2,3]naphthaleno, the resultant compound isanthracene. When R′ taken together with R² is [1,2]naphthaleno, theresultant compound is phenanthrene. In a preferred embodiment, thearyleno group is (C₅-C₁₄), with (C₅-C₁₀) being even more preferred.

[0075] “Arylaryl:” refers to a monovalent hydrocarbon radical derived bythe removal of one hydrogen atom from a single carbon atom of a ringsystem in which two or more identical or non-identical parent aromaticring systems are joined directly together by a single bond, where thenumber of such direct ring junctions is one less than the number ofparent aromatic ring systems involved. Typical arylaryl groups include,but are not limited to, biphenyl, triphenyl, phenyl-naphthyl,binaphthyl, biphenyl-naphthyl, and the like. When the number of carbonatoms comprising an arylaryl group is specified, the numbers refer tothe carbon atoms comprising each parent aromatic ring. For example,(C₁-C₁₄) arylaryl is an arylaryl group in which each aromatic ringcomprises from 5 to 14 carbons, e.g., biphenyl, triphenyl, binaphthyl,phenylnaphthyl, etc. Preferably, each parent aromatic ring system of anarylaryl group is independently a (C₅-C₁₄) aromatic, more preferably a(C₅-C₁₀) aromatic. Also preferred are arylaryl groups in which all ofthe parent aromatic ring systems are identical, e.g., biphenyl,triphenyl, binaphthyl, trinaphthyl, etc.

[0076] “Biaryl:” refers to an arylaryl radical having two identicalparent aromatic systems joined directly together by a single bond.Typical biaryl groups include, but are not limited to, biphenyl,binaphthyl, bianthracyl, and the like. Preferably, the aromatic ringsystems are (C₅-C₁₄) aromatic rings, more preferably (C₅-C₁₀) aromaticrings. A particularly preferred biaryl group is biphenyl.

[0077] “Arylalkyl:” refers to an acyclic alkyl radical in which one ofthe hydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylakenyl and/or arylalkynyl is used. In preferredembodiments, the arylalkyl group is (C₆-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₆) andthe aryl moiety is (C₅-C₁₄). In particularlypreferred embodiments thearylalkyl group is (C₆-C₁₃), e.g., the alkanyl, alkenyl or alkynylmoiety of the arylalkyl group is (C₁-C₃) and the aryl moiety is(C₅-C₁₀).

[0078] “Parent Heteroaromatic Ring System:” refers to a parent aromaticring system in which one or more carbon atoms (and any necessaryassociated hydrogen atoms) are each independently replaced with the sameor different heteroatom. Typical heteratoms to replace the carbon atomsinclude, but are not limited to, N, P, O, S, Si, etc. Specificallyincluded within the definition of “parent heteroaromatic ring systems”are fused ring systems in which one or more rings are aromatic and oneor more of the rings are saturated or unsaturated, such as, for example,arsindole, chromane, chromene, indole, indoline, xanthene, etc. Typicalparent heteroaromatic ring systems include, but are not limited to,arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, firan,imidazole, indazole, indole, indoline, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

[0079] “Heteroaryl:” refers to a monovalent heteroaromatic radicalderived by the removal of one hydrogen atom from a single atom of aparent heteroaromatic ring system. Typical heteroaryl groups include,but are not limited to, radicals derived from acridine, arsindole,carbazole, P-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isocbromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pynmidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike. In preferred embodiments, the heteroaryl group is a 5-14 memberedheteroaryl, with 5-10 membered heteroaryl being particularly preferred.The most preferred heteroaryl radicals are those derived from parentheteroaromatic ring systems in which any ring heteroatoms are nitrogens,such as imidazole, indole, indazole, isoindole, naphthyridine,pteridine, isoquinoline, phthalazine, purine, pyrazole, pyrazine,pyridazine, pyridine, pyrrole, quinazoline, quinoline, etc.

[0080] “Heteroaryldiyl:” refers to a divalent heteroaromatic radicalderived by the removal of one hydrogen atom from each of two differentatoms of a parent heteroaromatic ring system or ′ by the removal of twohydrogen atoms from a single atom of a parent heteroaromatic ringsystem. The two monovalent radical centers or each valency of the singledivalent center can form bonds with the same or different atom(s).Typical heteroaryldiyl groups include, but are not limited to, divalentradicals derived from acridine, arsindole, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In preferred embodiments,the heteroaryldiyl group is 5-14 membered heteroaryldiyl, with 5-10membered being particularly preferred. The most preferred heteroaryldiylgroups are divalent radicals derived from parent heteroaromatic ringsystems in which any ring heteroatoms are nitrogens, such as imidazole,indole, indazole, isoindole, naphthyridine, pteridine, isoquinoline,phthalazine, purine, pyrazole, pyrazine, pyridazine, pyridine, pyrrole,quinazoline, quinoline, etc.

[0081] “Heteroaryleno:” refers to a divalent bridge radical having twoadjacent monovalent radical centers derived by the removal of onehydrogen atom from each of two adjacent atoms of a parent heteroaromaticring system. Attaching a heteroaryleno bridge radical, e.g. pyridino, toa parent aromatic ring system, e.g. benzene, results in a fusedheteroaromatic ring system, e.g., quinoline. The bridge is assumed tohave the maximum number of non-cumulative double bonds consistent withits attachment to the resultant fused ring system. In order to avoiddouble-counting ring atoms, when a heteroaryleno substituent is formedby taking together two adjacent substituents on a structure thatincludes alternative substituents, the ring atoms of the heteroarylenobridge replace the bridging ring atoms of the structure. As an example,consider the following structure:

[0082] wherein:

[0083] R¹, when taken alone is hydrogen, or when taken together with R²is 5-14 membered heteroaryleno; and

[0084] R², when taken alone is hydrogen, or when taken together with R¹is 5-14 membered heteroaryleno;

[0085] When R¹ and R² are each hydrogen, the resultant compound isbenzene. When R¹ taken together with R² is a 6-membered heteroaryleno(e.g., pyridino), the resultant compound is isoquinoline, quinoline orquinolizine. When R′ taken together with R² is a 10-memberedheteroaryleno (e.g., isoquinoline), the resultant compound is, e.g.,acridine or phenanthridine. Typical heteroaryleno groups include, butare not limited to, acridino, carbazolo, β-carbolino, chromeno,cinnolino, furano, imidazolo, indazoleno, indoleno, indolizino,isobenzoflrano, isochromeno, isoindoleno, isoquinolino, isothiazoleno,isoxazoleno, naphthyridino, oxadiazoleno, oxazoleno, perimidino,phenanthridino, phenanthrolino, phenazino, phthalazino, pteridino,purino, pyrano, pyrazino, pyrazoleno, pyridazino, pyridino, pyrimidino,pyrroleno, pyrrolizino, quinazolino, quinolino, quinolizino,quinoxalino, tetrazoleno, thiadiazoleno, thiazoleno, thiopheno,triazoleno, xantheno, and the like. Where a specific connectivity isintended, the involved bridging atoms (of the heteroaryleno bridge) aredenoted in brackets, e.g., [1,2]pyridino, [2,3]pyridino, [3,4]pyridino,etc. Thus, in the above example, when R′ taken together with R² is[1,2]pyridino, the resultant compound is quinolizine. When R¹ takentogether with R² is [2,3]pyridino, the resultant compound is quinoline.When R¹ taken together with R² is [3,4]pyridino, the resultant compoundis isoquinoline. In preferred embodiments, the heteroaryleno group is5-14 membered heteroaryleno, with 5-10 membered being even morepreferred. The most preferred heteroaryleno radicals are those derivedfrom parent heteroaromatic ring systems in which any ring heteroatomsare nitrogens, such as imidazolo, indolo, indazolo, isoindolo,naphthyridino, pteridino, isoquinolino, phthalazino, purino, pyrazolo,pyrazino, pyridazino, pyridino, pyrrolo, quinazolino, quinolino, etc.

[0086] “Heteroaryl-Heteroaryl:” refers to a monovalent heteroaromaticradical derived by the removal of one hydrogen atom from a single atomof a ring system in which two or more identical or non-identical parentheteroaromatic ring systems are joined directly together by a singlebond, where the number of such direct ring junctions is one less thanthe number of parent heteroaromatic ring systems involved. Typicalheteroaryl-heteroaryl groups include, but are not limited to, bipyridyl,tripyridyl, pyridylpurinyl, bipurinyl, etc. When the number of ringatoms are specified, the numbers refer to the number of atoms comprisingeach parent heteroatomatic ring systems. For example, 5-14 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 14 atoms, e.g.,bipyridyl, tripyridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-14 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical. The most preferred heteroaryl-heteroaryl radicals are thosein which each heteroaryl group is derived from parent heteroaromaticring systems in which any ring heteroatoms are nitrogens, such asimidazole, indole, indazole, isoindole, naphthyridine, pteridine,isoquinoline, phthalazine, purine, pyrazole, pyrazine, pyridazine,pyridine, pyrrole, quinazoline, quinoline, etc.

[0087] “Biheteroaryl:” refers to a heteroaryl-heteroaryl radical havingtwo identical parent heteroaromatic ring systems joined directlytogether by a single bond. Typical biheteroaryl groups include, but arenot limited to, bipyridyl, bipurinyl, biquinolinyl, and the like.Preferably, the heteroaromatic ring systems are 5-14 memberedheteroaromatic rings, more preferably 5-10 membered heteroaromaticrings. The most preferred biheteroaryl radicals are those in which theheteroaryl groups are derived from a parent heteroaromatic ring systemin which any ring heteroatoms are nitrogens, such as biimidazolyl,biindolyl, biindazolyl, biisoindolyl, binaphthyridinyl, bipteridinyl,biisoquinolinyl, biphthalazinyl, bipurinyl, bipyrazolyl, bipyrazinyl,bipyridazinyl, bipyridinyl, bipyrrolyl, biquinazolinyl, biquinolinyl,etc.

[0088] “Heteroarylalkyl:” refers to an acyclic alkyl radical in whichone of the hydrogen atoms bonded to a carbon atom, typically a terminalor sp³ carbon atom, is replaced with a heteroaryl radical. Wherespecific alkyl moieties are intended, the nomenclatureheteroarylalkanyl, heteroarylakenyl and/or heterorylalkynyl is used. Inpreferred embodiments, the heteroarylalkyl group is a 6-20 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of theheteroarylalkyl is 1-6 membered and the heteroaryl moiety is a5-14-membered heteroaryl. Inparticularlypreferred embodiments, theheteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety is 1-3 membered and the heteroaryl moiety is a5-10 membered heteroaryl.

[0089] “Substituted:” refers to a radical in which one or more hydrogenatoms are each independently replaced with the same or differentsubstituent(s). Typical substituents include, but are not limited to,—X, —R, —O⁻, ═O, —OR, —O—OR, —SR, —S⁻, ═S, —NRR, ═NR, perhalo (C₁—C₆)alkyl, —CX₃, —CF₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃,—S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR,—C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR,where each X is independently a halogen (preferably —F or —Cl) and eachR is independently hydrogen, alkyl, alkanyl, alkenyl, alkynyl, aryl,arylalkyl, arylaryl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl, as defined herein. The actual substituentsubstituting any particular group will depend upon the identity of thegroup being substituted.

[0090] “Parent Xanthene Ring:” refers to a heteroaromatic ring system ofa type typically found in the xanthene class of fluorescent dyes (whichincludes rhodamine and fluorescein dyes, defined infra), i.e., aheteroaromatic ring system having the general structure:

[0091] In the parent xanthene ring depicted above, A¹ is —OH or —NH₂ andA² is ═O or ═NH₂ ⁺. When A¹ is —OH and A² is ═O, the parent xanthenering is a fluorescein-type parent xanthene ring, which is defined inmore detail, infra. When A¹ is —NH₂ and A² is ═NH₂ ⁺, the parentxanthene ring is a rhodamine-type parent xanthene ring, which is definedin more detail, infra. When A¹ is —NH₂ and A² is ═O, the parent xanthenering is a rhodol-type parent xanthene ring. In the parent xanthene ringdepicted above, one or both nitrogens of A¹ and A² (when present) and/orone or more of the carbon atoms at positions C1, C2, C4, C5, C7 and C8,can be independently substituted with a wide variety of the same ordifferent substituents, as is well known in the art. Typicalsubstituents include, but are not limited to, —X, —R, —OR, —SR, —NRR,perhalo (C₁-C₆) alkyl, —CX₃, —CF₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X,—C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and—C(NR)NRR, where each X is independently a halogen (preferably —F or—Cl) and each R is independently hydrogen, (C₁-C₆) alkyl, (C₁-C₆)alkanyl, (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, (C₆-C₂₆)arylalkyl, (C₅-C₂₀) arylaryl, heteroaryl, 6-26 membered heteroarylalkylor 5-20 membered heteroaryl-heteroaryl. Moreover, the C1 and C2substituents and/or the C7 and C8 substituents can be taken together toform substituted or unsubstituted (C₅-C₂₀) aryleno bridges. Generally,substituents groups which do not tend to quench the fluorescence of theparent xanthene ring are preferred, but in some embodiments quenchingsubstituents may be desirable. Substituents that tend to quenchfluorescence of parent xanthene rings are electron-withdrawing groups,such as —NO₂, —F, —Br, —CN and —CF₃.

[0092] When A¹ is —NH₂ and/or A² is ═NH₂, the xanthene nitrogens can beincluded in bridges involving the same nitrogen atom or adjacent carbonatoms, e.g., (C₁-C₁₂) alkyldiyl, (C₁-C₁₂) alkyleno, 2-12 memberedheteroalkyldiyl and/or 2-12 membered heteroalkyleno bridges.

[0093] Any of the substituents substituting carbons C1, C2, C4, C5, C7or C8 and/or the xanthene nitrogen atoms (when present) can be furthersubstituted with one or more of the same or different substituents,which are typically selected from the group consisting of —X, —R′, ═O,—OR′, —SR′, ═S, —NR′R′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO₂, ═N₂, —N₃, —NHOH, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —P(O)(O—)₂,—P(O)(OH)₂, —C(O)R′, —C(O)X, —C(S)R′, —C(S)X, —C(O)OR′, —C(O)O⁻,—C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′, —C(S)NR′R′ and —C(NR)NR′R′,where each X is independently a halogen (preferably —F or —Cl) and eachR′ is independently hydrogen, (C₁-C₆) alkyl, 2-6 membered heteroalkyl,(C₅-C₁₄) aryl or heteroaryl.

[0094] Exemplary parent xanthene rings include, but are not limited to,rhodamine-type parent xanthene rings and fluorescein-type parentxanthene rings, each of which is defined in more detail, infra.

[0095] “Rhodamine-Type Parent Xanthene Ring:” refers to a parentxanthene ring in which A¹ is —NH₂ and A² is ═NH₂ ⁺, i.e., a parentxanthene ring having the general structure:

[0096] In the rhodamine-type parent xanthene ring depicted above, one orboth nitrogens and/or one or more of the carbons at positions C1, C2,C4, C5, C7 or C8 can be independently substituted with a wide variety ofthe same or different substituents, as previously described for theparent xanthene rings. Exemplary rhodamine-type parent xanthene ringsinclude, but are not limited to, the xanthene rings of the rhodaminedyes described in U.S. Pat. No. 5,936,087; U.S. Pat. No. 5,750,409; U.S.Pat. No. 5,366,860; U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,840,999;U.S. Pat. No. 5,847,162; U.S. application Ser. No. 09/277,793, filedMar. 27, 1999; PCT Publication WO 97/36960; PCT Publication WO 99/27020;Sauer et al., 1995, J. Fluorescence 5(3):247-261; Arden-Jacob, 1993,Neue Lanwellige Xanthen-Farbstoffe für Fluoreszenzsonden und FarbstoffLaser, Verlag Shaker, Germany; and Lee et al., 1992, Nucl. Acids Res.20(10):2471-2483. Also included within the definition of “rhodamine-typeparent xanthene ring” are the extended-conjugation xanthene rings of theextended rhodamine dyes described in U.S. application Ser. No.09/325,243, filed Jun. 3, 1999.

[0097] “Fluorescein-Type Parent Xanthene Ring:” refers to a parentxanthene ring in which A¹ is —OH and A² is ═O, i.e., a parent xanthenering having the structure:

[0098] In the fluorescein-type parent xanthene ring depicted above, oneor more of the carbons at positions C1, C2, C4, C5, C7 or C8 can beindependently substituted with a wide variety of the sarne or differentsubstituents, as previously described for the parent xanthene rings.Exemplary fluorescein-type parent xanthene rings include, but are notlimited to, the xanthene rings of the fluorescein dyes described in U.S.Pat. No. 4,439,356; U.S. Pat. No. 4,481,136; U.S. Pat. No. 5,188,934;U.S. Pat. No. 5,654,442; U.S. Pat. No. 5,840,999; WO 99/16832;and EP 0050 684. Also included within the definition of “fluorescein-type parentxanthene ring” are the extended xanthene rings of the fluorescein dyesdescribed in U.S. Pat. No. 5,750,409 and U.S. Pat. No. 5,066,580.

[0099] “Xanthene Dye:” refers to a class of fluorescent dyes whichconsist of a parent xanthene ring substituted at the xanthene C-9 carbonwith a substituted phenyl ring or other, typically acyclic, substituent.Common substituted phenyl rings found in xanthene dyes include, e.g.,2-carboxyphenyl, dihalo-2-carboxyphenyl, tetrahalo-2-carboxyphenyl,2-ethoxycarbonylphenyl, dihalo-2-ethoxycarbonylphenyl andtetrahalo-2-ethoxycarbonylphenyl. Common acyclic substituents found inxanthene dyes include, e.g., carboxyethyl and perfluoroalkyl (eg.,tfifluoromethyl, pentafluoroethyl and heptafluoropropyl). Typicalxanthene dyes include the fluorescein dyes and the rhodamine dyes, whichare described in more detail, infra

[0100] “Rhodamine Dye:” refers to the subclass of xanthene dyes in whichthe xanthene ring is a rhodamine-type parent xanthene ring. Typicalrhodamine dyes include, but are not limited to, rhodamine B,5-carboxyrhodamine, rhodamnine X (ROX), 4,7-dichlororhodamine X (dROX),rhodamine 6G (R6G), rhodamine 110 (R110), 4,7-dichlororhodamine 110(dR110), tetramethyl rhodamine (TAMRA) and4,7-dichloro-tetramethylrhodamine (dTAMRA). Additional typical rhodaminedyes can be found, for example, in U.S. Pat. No. 5,936,087; U.S. Pat.No. 5,750,409; U.S. Pat. No. 5,366,860; U.S. Pat. No. 5,231,191; U.S.Pat. No. 5,840,999; U.S. Pat. No. 5,847,162; U.S. application Ser. No.09/038,191, filed Mar. 10, 1998; U.S. application Ser. No. 09/277,793,filed Mar. 27, 1999; U.S. application Ser. No. 09/325,243, filed Jun. 3,1999; PCT Publication WO 97/36960; PCT Publication WO 99/27020; Sauer etal., 1995, J. Fluorescence 5(3):247-261; Arden-Jacob, 1993, NeueLanwellige Xanthen-Farbstoffe für Fluoreszenzsonden und Farbstoff Laser,Verlag Shaker, Germany; and Lee et al., 1992, Nucl. Acids Res.20(10):2471-2483.

[0101] “Fluorescein Dye:” refers to the subclass of xanthene dyes inwhich the parent xanthene ring is a fluorescein-type parent xanthenering. Typical fluorescein dyes include, but are not limited to,5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM). Additionaltypical fluorescein dyes can be found, for example, in U.S. Pat. No.5,750,409; U.S. Pat. No. 5,066,580; U.S. Pat. No. 4,439,356; U.S. Pat.No. 4,481,136; U.S. Pat. No. 5,188,934; U.S. Pat. No. 5,654,442; U.S.Pat. No. 5,840,999; PCT publication WO 99/16832; EP 0 050 684; and U.S.application Ser. No. 08/942,067, filed Oct. 1, 1997.

[0102] 5.3 The Rhodamine Dye Compounds 5.3.1 The Compounds Per Se

[0103] The rhodamine dyes of the invention are generally rhodamine-typeparent xanthene rings substituted at the xanthene C9 position with a newbottom ring. The new bottom ring is a phenyl group which bears fivesubstituents: an ortho carboxyl or sulfonate group (or salts thereof);one to three aminopyridinium (Pyr⁺) groups; and one alkylthio, arylthioor heteroarylthio group. The Pyr⁺ groups, which may be the same ordifferent, are typically the same and are attached to the new bottomring via the pyridinium ring nitrogen. The alkylthio, arylthio orheteroarylthio group is attached to the new bottom ring via the sulfuratom. As previously described in the Summary section, theaminopyridinium and/or alkylthio, arylthio or heteroarylthio groups maybe substituted or unsubstituted. The rhodamine dyes may also include anoptional linking moiety, described in more detail, infra.

[0104] Currently available red-emitting fluorescent dyes, such asrhodamines and cyanines, suffer from undesirable water-solubility and/orphotostability characteristics. For example, due to their hydrophobicnature, most commercially-available rhodanine dyes are somewhatinsoluble in water. Red-emitting cyanine dyes such as Cy5, althoughwater-soluble, are photo unstable. Thus, available red-emittingrhodamine and cyanine dyes are not well-suited for many aqueous-basedbiological applications, such as cell staining or nucleic acidsequencing.

[0105] By virtue or their new bottom rings, the rhodamine dyes of theinvention overcome these limitations. While not intending to be bound byany particular theory, it is believed that the Pyr⁺ groups substitutingthe new bottom ring render the dyes highly water-soluble. Quiteimportantly, rhodamine dyes substituted with these new bottom ringsretain their characteristic photostability. Moreover, the new bottomring tends to shift the emissions spectral properties of the dyes to thered by about 5-30 run, as compared with corresponding rhodamines dyescomprising a conventional bottom ring. Thus, the rhodamine dyes of theinvention are ideally suited for use as water-soluble laser dyes and inaqueous-based biological applications such as cell staining and nucleicacid sequencing. Quite significantly, when used to label terminators innucleic acid sequencing applications, the new rhodamine dyes of theinvention do not effect obscuring impurities which electrophoreticallymigrate in the range of DNA sequencing fragments. Obscuring impuritiesare commonly observed with conventionally-labeled terminators and aregenerally thought to be caused by unincorporated labelled-terminators.As a consequence, sequencing data obtained with terminators labeled withthe new rhodamine dyes of the invention is typically much higher inquality than that obtained with conventional terminators. Sequencingreactions obtained with terminators labeled with the new rhodamine dyesof the present invention may require less sample purification, or “cleanup” than those employing conventional terminators.

[0106] The rhodamine dyes of the invention are generally compoundsaccording to structural formula (I):

[0107] including any associated counterions, wherein:

[0108] E is carboxylic acid, sulfonic acid, or a salt thereof;

[0109] Y is a rhodamine-type parent xanthene ring connected to theillustrated fully substituted phenyl ring at the C-9 carbon;

[0110] each Pyr⁺ is independently a substituted or unsubstitutedaminopyridiniun group connected to the illustrated phenyl ring via thepyridinium ring nitrogen;

[0111] S is sulfur;

[0112] n is one, two, or three; and

[0113] Z a substituted or unsubstituted (C₁-C₁₂) alkyl, (C₅-C₁₄) aryl orheteroaryl.

[0114] The invention is based, in part, on the discovery that replacingthe ring or substituent attached to the xanthene C-9 carbon ofconventional rhodamine dyes with the new bottom rings described hereinyields rhodamine dyes having superior water-solubility, photo stabilityand/or excitation and emission spectral properties. As a consequence,those of skill in the art will recognized that in the rhodamine dyes ofstructural formula (I), rhodamine-type parent xanthene ring Y can bederived from virtually any fluorescent rhodamine dye that is now knownor that will be later developed. Exemplary rhodamine-type parentxanthene rings that can comprise Y include, but are not limited to, anyof the substituted or unsubstituted xanthene rings of the rhodamine dyesdescribed in U.S. Pat. No. 5,936,087; U.S. Pat. No. 5,750,409; U.S. Pat.No. 5,366,860; U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,840,999; U.S.Pat. No. 5,847,162; U.S. application Ser. No. 09/038,191, filed Mar. 10,1998; U.S. application Ser. No. 09/277,793, filed Mar. 27, 1999; U.S.application Ser. No. 09/325,243, filed Jun. 3, 1999; PCT Publication WO97/36960; PCT Publication WO 99/27020; Sauer et al., 1995, J.Fluorescence 5(3):247-261; Arden-Jacob, 1993, Neue LanwelligeWanthen-Farbstoffe für Fluoreszenzsonden und Farbstoff Laser, VerlagShaker, Germany; and Lee et al, 1992, Nucl. Acids Res. 20(10):2471-2483,the disclosures of which are incorporated herein by reference.

[0115] While the dyes of structural formula (I) are useful in virtuallyany aqueous-based applications employing red-emitting fluorescent dyes,such as, for example, as water-soluble laser dyes, rhodamines accordingto structural formula (I) which incorporate one or more optional linkingmoieties are particularly useful, as they can be specifically and/orpermanently conjugated to other compounds and/or substances so as tolabel the compounds or substances for subsequent detection. The linkingmoieties are attached to the rhodamine-type parent xanthene ring, eitherat a xanthene nitrogen, the xanthene C4 carbon, and/or the Zsubstituent. Thus, preferred rhodamine dyes of the invention arecompounds according to structural formula (1) in which the C4 carbonatom of Y, one or both nitrogen atoms of Y and/or substituent Z aresubstituted with a linking moiety of the formula —L—, wherein L is abond or linker. When the dye includes multiple linkers L, each may bethe same or different.

[0116] The nature of linker L will depend upon the particularapplication, point of attachment and type of conjugation desired. LinkerL may be attached directly to the dye, or it may be spaced away from thedye through one or more intervening atoms that serve as a linker. In theformer embodiment, L represents a bond. In the latter embodiment, Lrepresents a linker of more than one atom. The linker can be hydrophilicor hydrophobic, long or short, rigid, semirigid or flexible, dependingupon the particular application. The linker can be optionallysubstituted with one or more substituents or one or more additionallinking groups, which may be the same or different, thereby providing a“polyvalent” linking moiety capable of conjugating with multiplemolecules or substances. Preferably, however, linker L does not includesuch additional substituents or linking groups.

[0117] A wide variety of linkers L comprised of stable bonds are knownin the art, and include by way of example and not limitation,alkyldiyls, substituted alkyldiyls, alkylenos, substituted alkylenos,heteroalkyldiyls, substituted heteroalkyldiyls, heteroalkylenos,substituted heteroalkylenos, acyclic heteroatomic bridges, aryldiyls,substituted aryldiyls, arylaryldiyls, substituted arylaryldiyls,arylalkyldiyls, substituted arylalkyldiyls, heteroaryldiyls, substitutedheteroaryldiyls, heteroaryl-heteroaryldiyls, substitutedheteroaryl-heteroaryldiyls, heteroarylalkyldiyls, substitutedheteroarylalkyldiyls, heteroaryl-heteroalkyldiyls, substitutedheteroaryl-heteroalkyldiyls, and the like. Thus, linker L may includesingle, double, triple or aromatic carbon-carbon bonds,nitrogen-nitrogen bonds, carbon-nitrogen, carbon-oxygen bonds and/orcarbon-sulfur bonds, and may therefore include functionalities such ascarbonyls, ethers, thioethers, carboxamides, sulfonamides, ureas,urethanes, hydrazines, etc. In one embodiment, linker L has from 1-20non-hydrogen atoms selected from the group consisting of C, N, O, and Sand is composed of any combination of ether, thioether, amine, ester,carboxamide, sulfonamides, hydrazide, aromatic and heteroaromatic bonds.

[0118] Choosing a linker having properties suitable for a particularapplication is within the capabilities of those having skill in the art.For example, where a rigid linker is desired, L may be a rigidpolypeptide such as polyproline, a rigid polyunsaturated alkyldiyl or anaryldiyl, biaryldiyl, arylarydiyl, arylalkyldiyl, heteroaryldiyl,biheteroaryldiyl, heteroarylalkyldiyl, heteroaryl-heteroaryldiyl, etc.Where a flexible linker is desired, L may be a flexible polypeptide suchas polyglycine or a flexible saturated alkanyldiyl or heteroalkanyldiyl.Hydrophilic linkers may be, for example, polyalcohols or polyethers suchas polyalkyleneglycols. Hydrophobic linkers may be, for example,alkyldiyls or aryldiyls.

[0119] Linkers suitable for use in most biological applications include(C₁-C₁₂) alkyldiyls, particularly alkanylenos such as methano (—CH₂—),ethano (—CH₂—CH₂—), propano (—CH₂—CH₂—CH₂—), butano (—CH₂—CH₂—CH₂—CH₂—),pentano (—CH₂CH₂—CH₂—CH₂—CH₂) and hexano (—CH₂—CH₂—CH₂—CH₂—CH₂CH₂—);(C₅-C₂₀) arYldiyls, particularlyphena-1,3-diyl

[0120] phena-1,4-diyl

[0121] naphtha-2,6-diyl

[0122] and naphtha-2,7-diyl

[0123] and (C₆-C₂₆) arylalkyldiyls, particularly those having thestructural formula —CH₂)_(i)φ- or —(CH₂)_(i)-ψl, where each i isindependently an integer from 1 to 6, φ is phenyldiyl (especiallyphena-1,3-diyl or phena-1,4-diyl) and ψ is naphthyldiyl (especiallynaphtha-2,6-diyl or naphtha-2,7-diyl). Rigid linkers that are suitablefor attaching the dyes of the invention to one another or to other dyesto create energy-transfer dye pairs (described in more detail, infra)include —(CH₂)_(i)—NR″—C(O)-φ and —(CH₂)_(i)—NR″—C(O)-ψ, where i, R″, φand ψ are as previously defined. Particularly preferred are thoselinkers in which i is 1. Analogs of all of these linkers L containingone or more heteroatomic groups, particularly those selected from thegroup consisting of O, S, N and NR″, where R″ is hydrogen or (C₁-C₆)alkyl, can also be conveniently used to space linkers from the rhodaminedyes of the invention. Linkers L tailored to specific applications arediscussed in more detail, infra.

[0124] Rhodamine dyes including a linking moiety can be conjugated to avariety of different molecules and substances using a plethora ofdifferent conjugation means. For example, the conjugation can bemediated via hydrophobic interactions, ionic attraction, through the useof pairs of specific binding molecules such as biotin andavidin/streptavidin or through covalent attachment. When conjugation viahydrophobic interactions is desired, linker L is a hydrophobic moietythat is capable of forming hydrophobic interactions with a hydrophobicmoiety on the molecule or substance to be conjugated. Typicalhydrophobic moieties include, but are not limited to, unsubstituted andsubstituted aryl, arylalkyl, arylaryl, heteroaryl, heteroarylaklyl andheteroaryl-heteroaryl groups. When the hydrophobic moiety issubstituted, the substituents are preferably nonpolar, more preferablyhydrophobic. Suitable hydrophobic moieties for forming non-covalentconjugates will be apparent to those of skill in the art.

[0125] When conjugation via ionic attraction is desired, L is a chargedmoiety having a net charge of a polarity opposite to a net charge on themolecule or substance to be conjugated. Typical charged moietiesinclude, by way of example and not limitation, quaternary ammoniums,carboxylates and sulfonates, including salts thereof. A variety ofcyclic quaternary ammoniums that are suitable for use in linkers aredescribed in U.S. Pat. No. 5,863,753 (see, e.g., Cols. 8-9), thedisclosure of which is incorporated herein by reference.

[0126] When conjugation via pairs of specific binding molecules such asbiotin and avidin/streptavidin is desired, L will constitute one memberof the binding pair. The molecule or substance to be conjugated willbear the other member of the binding pair. Where one of the members ofthe specific binding pair is a small molecule, such as biotin or ahormone, that member preferably comprises L. A variety of biotinscapable of being covalently linked to reactive functional groups such asamines are commercially available (e.g., Molecular Probes, Eugene,Oreg.). These biotins can be incorporated into the dyes of the inventionto yield biotin-labeled dyes suitable for non-covalent conjugation to avariety of avidin/streptavidin-labeled molecules or substances.

[0127] Preferably, L is capable of mediating conjugation via covalentattachment. In this preferred embodiment, L bears a reactive functionalgroup (R_(X) ). Covalent conjugates are obtained by reacting a rhodaminedye of the invention including a reactive group R_(X) with a molecule orsubstance that contains, or is modified to contain, one or morefunctional groups F_(x) that are complementary to reactive group R_(X) .

[0128] The exact identities of R_(X) and F_(x) will depend upon thenature of the desired covalent linkage and the chemistry used to formthe covalent linkage. Generally, reactive group R_(X) is a functionalgroup that is capable of reacting with a complementary functional groupF_(x) under specified reaction conditions to form a covalent linkage.However, those of skill in the art will recognize that a variety offunctional groups that are typically unreactive under certain reactionconditions can be activated to become reactive. Groups that can beactivated to become reactive include, e.g., carboxylic acids and esters,including salts thereof. Such groups are referred to herein as“activatable precursors” and are specifically intended to be includedwithin the expression “reactive group.”

[0129] Pairs of reactive groups R_(X) and complementary groups F_(x)suitable for forming covalent linkages with one another under a varietyof different reaction conditions are well-known. Any of thesecomplementary pairs of groups can be used to covalently conjugate therhodamine dyes of the invention to other compounds or substances. In oneconvenient embodiment, reactive group R_(X) and complementary functionalgroup F_(x) comprise complementary electrophiles and nucleophiles (ortheir respective activatable precursors). In another convenientembodiment, reactive group R_(X) is a photoactivatable group thatbecomes chemically reactive only after illumination with light of anappropriate wavelength and complementary functional group F_(x) is agroup capable forming a covalent linkage with the chemically reactivespecies. Such photoactivatable groups can be conveniently used to photocross-link the rhodamine dyes of the invention to other molecules and/orsubstances.

[0130] As understood in the art, “activated esters” generally have theformula —C(O)Ω, where Ω is a good leaving group. Exemplary good leavinggroups include, by way of example and not limitation: oxysuccinimidyl;N-succinimidyl; oxysulfosuccinimidyl; 1-oxybenzotriazolyl; and —OR^(a),where R^(a) is selected from the group consisting of (C₄-C₂₀) cycloalkyl(e.g. cyclohexyl), heterocycloalkyl, (C₅-C₂₀) aryl, (C₅-C₂₀) arylsubstituted with one or more of the same or differentelectron-withdrawing groups (e.g., —NO₂, —F, —Cl, —CN, —CF₃, etc.),heteroaryl, and heteroaryl substituted with one or more of the same ordifferent electron-withdrawing groups, n-dialkylaminoalkyls (e.g.,3-dimethylaminopropyl) and N-morpholinomethyl, or R^(a) is used to forman anhydride of the formula —OCOR^(b) or —OCNR^(b)NHR^(c), where R^(b)and R^(c) are each independently selected form the group consisting of(C₁-C₆) alkyl, (C₁-C₆) perhaloalkyl, (C₁-C₆) perfluoroalkyl and (C₁-C₆)alkoxy. A preferred activated ester is NHS ester.

[0131] Exemplary photoactivatable groups suitable for conjugation vialight-activated cross-linking include, but are not limited to, azido(—N₃), 4-azido-phenyl and 2-nitro-4-azido-phenyl. Conjugation usingphotoactivatable groups typically involves illuminating a mixturecomprising the photoactivatable dyes and the molecule or substance to beconjugated, followed by separation of unreacted dyes and byproducts.

[0132] As will be recognized by those of skill in the art, reactivegroup R_(x) can comprise any electrophilic, nucleophilic orphotoactivatable groups. The selection of reactive group R_(x) used tocovalently conjugate the rhodamine dyes of the invention to the othermolecule or substance typically depends upon the identity of thecomplementary functional group F_(x) on the molecule or substance to beconjugated. The types of complementary functional groups typicallypresent on molecules or substances to be conjugated include, but are notlimited to, amines, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, mono- anddisubstituted amines, halides, epoxides, sulfonate esters, carboxylicacids or carboxylates, or a combination of these groups. A single typeof complementary functional group may be available on the molecule orsubstance (which is typical for polysaccharides), or a variety ofdifferent complementary functional groups may be available (e.g. amines,thiols, alcohols, phenols), which is typical for proteins. The moleculeor substance may be conjugated to more than one rhodamine dye, which maybe the same or different. Although some selectivity can be obtained bycarefully controlling the reaction conditions, selectivity ofconjugation is best obtained by appropriate choice of reactive groupR_(x) in light of the available complementary functional group(s) F_(x).In instances where the molecule or substance to be conjugated does notcontain available complementary functional group(s) F_(x), it can bemodified to contain such groups using any of a variety of standardtechniques.

[0133] In a preferred embodiment, reactive group R_(x) is a group thatreacts with, or that can be readily activated to react with, an amine, athiol or an alcohol. In a particularly preferred embodiment, one ofreactive group R_(x) or complementary functional group F_(x) is acarboxylic acid (or a salt thereof) or an activated ester, mostpreferably a N-hydroxysuccinimidyl (NHS) ester, and the other is anamine, preferably a primary amine. The NHS ester may be convenientlyobtained by reacting a rhodamine dye of the invention including acarboxylic acid reactive group R_(x) with N-hydroxysuccinimide in thepresence of an activating agent (e.g., dicyclohexylcarbodiimide)according to known methods.

[0134] For a discussion of the various reactive groups R_(x) andrespective complementary functional groups F_(x) that can beconveniently used to covalently conjugate the rhodamine dyes of theinvention to a variety of biological and other molecules or substances,as well as reaction conditions under which the conjugation reactions canbe carried out, see Haugland, 1996, Molecular Probes Handbook ofFluorescent Probes and Research Chemicals, Molecular Probes, Inc.;Brinkley, 1992, Bioconjugate Chem. 3:2 and Garman, 1997, Non-RadioactiveLabelling: A Practical Approach, Academic Press, London, as well as thereferences cited in all of the above.

[0135] In one illustrative embodiment, rhodamine dyes of the inventionwhich are capable of being covalently conjugated to other compoundsand/or substances are compounds according to structural formula (Ia):

[0136] including any associated counter ions, wherein:

[0137] E, Y, Pyr⁺ and S are as previously defined for structural formula(I);

[0138] Z¹ is a substituted or unsubstituted (C₁-C₁₂) alkyldiyl, (C₁-C₁₄)or heteroaryldiyl;

[0139] L is a bond or a linker as previously described;

[0140] R_(x) is a reactive group as previously described; and

[0141] n is one, two, or three.

[0142] In another illustrative embodiment, rhodamine dyes of theinvention which are capable of being covalently conjugated to othercompounds and/or substances are compounds according to structuralformula (Ib):

[0143] including any associated counterions, wherein:

[0144] E, Y, Pyr⁺, S and Z are as previously defined for structuralformula (I);

[0145] L and R_(x) are as previously defined for structural formula(Ia), where L is connected to the C4 carbon atom or a nitrogen atom ofY; and

[0146] n is one, two, or three.

[0147] While not intending to be bound by any particular theory, it isbelieved that the Pyr⁺ groups on the new bottom rings in the compoundsof structural formulae (I), (Ia) and (Ib) account for thewater-solubility of the rhodamine dyes of the invention.

[0148] As will be illustrated more thoroughly below, the Pyr⁺ groups areintroduced into the rhodamine dyes of the invention by displacingfluorine atoms of the corresponding tetrafluororhodamine precursor withthe desired aminopyridine reactant, e.g. Scheme (I), using a reactionsimilar to that described in Weiss, R. et al, 1995, Angew. Chem. Int.Ed. Engl. 34:1319-21; and Koch, A. et al, 1993, Jour. Org. Chem.58:1409-14. As a consequence, unless mixtures of different aminopyridinereactants are used in the displacement reaction, all of the Pyr+groupson the new bottom phenyl rings are identical. According to the reaction,the Pyr⁺ groups are attached to the new bottom phenyl ring via thepyridinium ring nitrogen.

[0149] The pyridinium ring carbons may be independently substituted witha wide variety of the same or different substituents. The desiredsubstituents are introduced as substituents on the aminopyridinereactants. These carbons may be substituted with virtually any group,with one caveat: in order to avoid deleteriously affecting thedisplacement reaction, the pyridine ring carbons should not be directlysubstituted with electron-withdrawing groups, e.g., —F, —Cl, —CF₃, —NO₂,—CN, —N₃, etc. However, such electron-withdrawing groups can be includedon the substituents, as long as it is not attached directly to apyridine ring carbon. For example, while a pyridine ring carbon shouldnot be directly substituted with —F, —Cl or —CF₃, it may be substitutedwith other, less electronegative or electron-withdrawing, haloalkyls(e.g. —CH₂—CH₂F). Identifying substituents which are suitablynon-electron-withdrawing is within the capabilities of those havingskill in the art. Typical groups useful for substituting the pyridiniumring carbons include, but are not limited to, —R, —OR, —SR, —NRR,—S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR,—C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR,where each R is independently hydrogen, (C₁-C₆) alkyl or heteroalkyl,(C₅-C₁₄) aryl or beteroaryl. The R groups may be further substitutedwith one or more of the same or different substituents, which aretypically selected from the group consisting of —X, —R′, ═O, —OR′, —SR′,═S, —NR′R′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂,—N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —C(O)R′, —C(O)X, —C(S)R′, —C(S)X,—C(O)OR′, —C(O)O—, —C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′, —C(S)NR′R′and —C(NR)NR′R′, where each X is independently a halogen (preferably —For —Cl) and each R′ is independently hydrogen, (C₁-C₆) alkyl, 2-6membered heteroalkyl, (C₁-C₁₄) aryl or heteroaryl. Preferably, thepyridinium ring carbons are unsubstituted. When substituted, the mostpreferred substituents are the same or different (C₁-C₆) alkyls.Preferably, the pyrdidiium carbons are unsubstituted. When substituted,the substituents are preferably the same or different (C₁-C₆) alkyls.

[0150] The amino groups of the Pyr⁺ substituents may be a primary,secondary or tertiary amino group, but is typically a tertiary amino.The nitrogen substituents, R, are typically the same or different(C₁-C₆) alkyl or heteroalkyl. The alkyl or heteroalkyl can be furthersubstituted with one or more of the same or different groups, aspreviously described for R, above.

[0151] Alternatively, the nitrogen atom may be included in a ringstructure having from 2 to 5 ring atoms. The ring may contain, inaddition to the amino nitrogen atom, one or more of the same ordifferent heteroatoms, which are typically selected from the groupconsisting of 0, S and N. The ring atoms can be further substituted withany of the previously described substituent groups. Preferably, theamino group is dimethylamino or morpholino.

[0152] In the compounds of structural formulae (I), (Ia) and (Ib), the Zor Z¹ substituent may also be substituted or unsubstituted, but ispreferably unsubstituted. When Z is an alkyl or Z¹ is an alkyldiyl,virtually any group can be used to substitute Z or Z¹. Typicalsubstituents include, but are not limited to, —X, —R, ═O, —OR, —SR, ═S,—NRR, ═NR, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃,—S(O)2O—, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR,—C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR,where each X is independently a halogen (preferably —F or —Cl) and eachR is independently hydrogen, (C₁-C₆) alkyl, 2-6 membered heteroalkyl,(C₅-C₁₄) aryl or heteroaryl. The R groups may be further substitutedwith one or more of the same or different substituents, which aretypically selected from the group consisting of —X, —R′, ═O, —OR′, —SR′,═S, —NR′R′, ═NR′, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂,—N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R′, —C(O)R′, —C(O)X, —C(S)R′, —C(S)X,—C(O)OR′, —C(O)O, —C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′, —C(S)NR′R′and —C(NR)NR′R′, where each X is independently a halogen (preferably —For —Cl) and each R′ is independently hydrogen, (C₁-C₆) alkyl, 2-6membered heteoralkyl, (C₁-C₁₄) aryl or heteroaryl.

[0153] However, referring to Scheme (I), it has been observed that thedisplacement reaction does not proceed efficiently with arylthio andheteroarylthio compounds 108 that are substituted at the aromatic ringatoms with halogen groups. Thus, when Z is an aryl or heteroaryl and/orZ¹ is an aryldiyl or heteroaryldiyl, the aromatic ring atoms should notbe substituted directly with halogens. However, as described above forthe pyridinium ring carbon substituents, the halogens can be included ina substituent (e.g., a haloalkyl), so long as the substituent is notsufficiently electronegative to disrupt the displacement reaction. Thus,with the exception of halogen, groups to substitute aryl, heteroaryl,aryldiyl and heteroaryldiyl Z and Z¹ groups are typically any of thegroups described above for when Z is an alkyl or Z¹ is an alkyldiyl.

[0154] The rhodamine dyes of the invention will now be more fullydescribed by reference to various preferred embodiments. In onepreferred embodiment, the rhodamine dyes of the invention are compoundsaccording to structural formulae (I), (Ia) and (Ib) in which each Pyr⁺is the same and has the structure:

[0155] including any associated counter ions, wherein:

[0156] R²² is selected from the group consisting of hydrogen and (C₁-C₆)alkyl;

[0157] R²³ is selected from the group consisting of hydrogen and (C₁-C₆)alkyl;

[0158] R²⁴, when taken alone, is selected from the group consisting of(C₁-C₆) alkyl, or when taken together with R^(24′) is (C₄-C₁₀)alkyldiyl, (C₄-C₆) alkyleno, heteroalkyldiyl or heteroalkyleno;

[0159] R^(24′), when taken alone, is selected from the group consistingof (C₁-C₆) alkyl, or when taken together with R²⁴ is (C₄-C₁₀) alkyldiyl,(C₄-C₆) alkyleno, heteroalkyldiyl or heteroalkyleno;

[0160] R²⁵ is selected from the group consisting of hydrogen and (C₁-C₆)alkyl; and

[0161] R²⁶ is selected from the group consisting of hydrogen and (C₁-C₆)alkyl.

[0162] In the Pyr⁺ substituents of structural formula (P.1), the dashedline at the pyridinium ring nitrogen indicates the point of attachmentto the phenyl ring in the compounds of structural formulae (I), (Ia)and/or (Ib). Preferred Pyr⁺ substituents according to structural formula(P.1) are those in which R²², R²³, R²⁵ and R²⁶ are each hydrogen and/orin which R²⁴ and R^(24′), when taken alone, are each the same (C₁-C₆)alkyl or, when taken together are a 4-6 membered heteroalkyleno having asingle oxygen heteroatom. Particularly preferred Pyr⁺ substituentsaccording to structural formula (P.1) are 4-(dimethylamino)pyridiniumand 4-(morpholino)pyridinium.

[0163] In another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formulae (I) and (Ib) inwhich Z is selected from the group consisting of (C₁-C₁₂) alkyl (C₁-C₁₂)alkyl substituted with one or more of the same or different W¹ groups;(C₅-C₁₄) aryl, (C₅-C₁₄) aryl substituted with one or more of the same ordifferent W² groups, 5-14 membered heteroaiyl or heteroarylindependently substituted with one or more of the same or different W²groups, wherein:

[0164] each W¹ is independently selected from the group consisting of—X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS,—NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X,—C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR,—C(S)NRR and —C(NR)NRR, where each X is independently a halogen(preferably —F or —Cl) and each R is independently hydrogen or (C₁-C₆)alkyl; and

[0165] each W² is independently selected from the group consisting of—R, —OR, —SR, —NRR, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, —N₃,—S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR,—C(O)O, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR, Ris as previously defined for W¹.

[0166] Preferably, Z is unsubstituted. However, when Z is a substituted(C₁-C₁₄) aryl or a substituted heteroaryl, the most preferredsubstituents are those that are not electron-withdrawing. The mostpreferred heteroaryl groups, whether substituted or unsubstituted, arethose in which any heteroatoms are nitrogens. Especially preferredamongst these preferred heteroaryls are pyridinyl and purinyl. The mostpreferred aryl groups, whether substituted or unsubstituted are phenyland naphthyl.

[0167] In another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ia) in which Z¹is selected from the group consisting of (C₁-C₁₂) alkyldiyl, (C₁-C₁₂)alkyldiyl substituted with one or more of the same or different W¹groups; (C₅-C₁₄) aryldiyl, (C₅-C₁₄) aryldiyl substituted with one ormore of the same or different W² groups, 5-14 membered heteroaryldiyl orheteroaryldiyl independently substituted with one or more of the same ordifferent W² groups, wherein W¹ and W² are as defined above.

[0168] Preferably, Z¹ is unsubstituted. However, when Z¹ is asubstituted (C₅-C₁₄) aryldiyl or a substituted heteroaryldiyl, the mostpreferred substituents are those that are not electron-withdrawing. Themost preferred heteroaryldiyl groups, whether substituted orunsubstituted, are those in which any heteroatoms are nitrogens.Especially preferred amongst these preferred heteroaryldiyls arepyridindiyl and purindiyl. The most preferred aryldiyl groups, whethersubstituted or unsubstituted are phendiyl and naphthadiyl, especiallyphena-1,3-diyl, phena-1,4-diyl, naphtha-2,6-diyl and naphtha-2,7-diyl.

[0169] In yet another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ia) in whichrhodamine-type parent xanthene ring is a compound according tostructural formula (Y-1):

[0170] including any associated counterions, wherein:

[0171] R¹ and R² are each independently selected from the groupconsisting of hydrogen and (C₁-C₆) alkyl;

[0172] R³, when taken alone, is selected from the group consisting ofhydrogen, (C₁-C₆) alkyl (C₅-C₁₄) aryl and (C₅-C₁₄) arylaryl, or whentaken together with R³ is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, or whentaken together with Rl or R⁴ is (C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

[0173] R³′, when taken alone, is selected from the group consisting ofhydrogen, (C₁-C₆) alkyl, (C₅-C₁₄) aryl and (C₅-C,₄) arylaryl, or whentaken together with R³ is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, or whentaken together with R² or R⁴ is (C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

[0174] R⁴, when taken alone, is selected from the group consisting ofhydrogen and (C₁-C₆) alkyl, or when taken together with R³ or R³ is(C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

[0175] R⁵, when taken alone, is selected from the group consisting ofhydrogen and (C₁-C₆) alkyl, or when taken together with R⁶ or R⁶ is(C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

[0176] R⁶, when taken alone, is selected from the group consisting ofhydrogen, (C₁-C₆) alkyl, (C₁-C₁₄) aryl and (C₅-C₁₄) arylaryl, or whentaken together with R⁶′ is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, orwhen taken together with R⁵ or R⁷ is (C₂-C₆) alkyldiyl or (C₂-C₆)alkyleno;

[0177] R^(6′), when taken alone, is selected from the group consistingof hydrogen, (C₁-C₆) alkyl, (C₅-C₁₄) aryl and (C₅-C₁₄) arylaryl, or whentaken together with R⁶ is (C₄-C₆) alkyldiyl or (C₄-C₆) alkyleno, or whentaken together with R⁵ or R⁷is(C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

[0178] R⁷, when taken alone, is selected from the group consisting ofhydrogen and (C₁-C₆) alkyl, or when taken together with R⁶ or R⁶′ is(C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno;

[0179] R⁸, when taken alone, is selected from the group consisting ofhydrogen and (C₁-C₆) alkyl; and

[0180] R⁹ indicates the point of attachment to phenyl bottom ring.

[0181] In another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ia) in whichrhodamine-type parent xanthene ring is a compound according tostructural formula (Y-2), (Y-3) or (Y-4):

[0182] including any associated counterions, wherein:

[0183] R¹, R³, R⁴, R⁵, R⁶, R⁸ and R⁹ are as previously defined forstructural formula (Y-1); and

[0184] R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ areeach independently selected from the group consisting of hydrogen and(C₁-C₆) alkyl, or R¹⁰, R¹¹, R¹² and R¹³ taken together are (C₅-C₁₄)aryleno or (C₅-C₁₄) aryleno substituted with one or more of the same ordifferent (C₁-C₆) alkyl, or R¹⁸, R¹⁹, R²⁰ and R²¹ taken together are(C₅-C₁₄) aryleno or (C₅-C₁₄) aryleno substituted with one or more of thesame or different (C₁-C₆) alkyl.

[0185] The dashed bonds in structural formulae (Y-2) and (Y-4) indicatebonds which can each, independently of one another, be a single or adouble bond. When these bonds are double bonds, one of R¹⁰ or R¹¹ andone of R¹² or R¹³ are taken together to form a bond and one of R¹⁸ orR¹⁹ and one of R²⁰ or R²¹ are taken together to form a bond. When thesebond are single bonds, the R¹⁰, R¹¹, R¹², R¹³, R¹⁸, R¹⁹, R²⁰ and R²¹substituents are as defined above.

[0186] In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ib) in whichrhodamine-type parent xanthene ring Y is selected from the groupconsisting of (Y-1), (Y-2), (Y-3) and (Y-4), where R′, R², R³, R⁵, R⁶,R⁶, R⁷ R⁸, R⁹ R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ andR²¹ are as previously defined, and either R³ or R⁴ indicates the pointof attachment of substituent L. When substituent L is attached toR^(3′), R⁴is as previously defined. When substitutent L is attached toR⁴, R^(3′) is as previously defined.

[0187] In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formulae (Ia) and (Ib)in which Y is selected from the group consisting of (Y-1), (Y-2), (Y-3)and (Y-4), and further in which:

[0188] any alkyl groups are alkanyls selected from the group consistingof methanyl, ethanyl and propanyl;

[0189] any aryl groups are phenyl or naphthyl;

[0190] any arylaryl groups are biphenyl;

[0191] any alkyldiyl or alkyleno bridges formed by taking R² togetherwith R³ or R³ are alkanyldiyls or alkanos, especially those selectedfrom the group consisting of ethano, propano, 1,1-dimethylethano,1,1-dimethylpropano and 1,1,3-trimethylpropano;

[0192] any alkyldiyl or alkyleno bridges formed by taking R³ togetherwith R^(3′) are alkanyldiyl or alkano, especially butano;

[0193] any alkyldiyl or alkyleno bridges formed by taking R⁴ togetherwith and R³ or R^(3′) are alkanyldiyls or alkanos, especially thoseselected from the group consisting of ethano, propano,1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano;

[0194] any alkyldiyl or alkyleno bridges formed by taking R⁵ togetherwith R⁶ or R^(6′) are alkanyldiyls or alkanos, especially those selectedfrom the group consisting of ethano, propano, 1,1-dimethylethano,1,1-dimethylpropano and 1,1,3-trimethylpropano;

[0195] any alkyldiyl or alkyleno bridges formed by taking R⁶ togetherwith R⁶ are alkanyldiyl or alkano, especially butano;

[0196] any alkyldiyl or alkyleno bridges formed by taking R⁷ togetherwith R⁶ or R^(6′) are alkanyldiyls or alkanos, especially those selectedfrom the group consisting of ethano, propano, 1,1-dimethylethano,1,1-dimethylpropano and 1,1,3-trimethylpropano;

[0197] any aryleno bridges formed by taking R¹⁰, R¹¹, R¹² and R¹³together are benzo; and

[0198] any aryleno bridges formed by taking R¹⁸, R¹⁹, R²⁰ and R²¹together are benzo.

[0199] In yet another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (la) in whichrhodamine-type parent xanthene ring Y is selected from the followinggroup of compounds, where R⁹ is as previously defined for structuralformula (Y-1):

[0200] In yet another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ia) in which:

[0201] Y is selected from the group consisting of (Y-1), (Y-2), (Y-3),(Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a), (Y-31a),(Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-42a), (Y-43a), (Y-44a),(Y-45a), and (Y-46a);

[0202] Z¹ is selected from the group consisting of (C₁-C₁₂) alkyleno,(C₁-C₁₂) alkano, (C₅-C₁₀) aryldiyl, phenyldiyl, phena-1,4-diyl,naphthadiyl, naphtha-2,6-diyl and heteroaryldiyl, pyridindiyl andpurindiyl.

[0203] In yet another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ia) in which:

[0204] L is a bond; and

[0205] Y is selected from the group consisting of (Y-1),(Y-2), (Y-3),(Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a), (Y-31a),(Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-42a), (Y-43a), (Y-44a),(Y-45a), and (Y-46a);

[0206] In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (la) in which:

[0207] R_(x) is any of the electrophilic or nucleophilic groups; and

[0208] Y is selected from the group consisting of (Y-1), (Y-2), (Y-3),(Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a), (Y-31a),(Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-42a), (Y-43a), (Y-44a),(Y-45a), and (Y-46a);

[0209] In yet another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ib) in which Yis selected from the group consisting of (Y-1),(Y-2), (Y-3),(Y-4),(Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a), (Y-31a), (Y-34a),(Y-35a), (Y-36a), (Y-39a), (Y-41 a), (Y-43a) and (Y-46a), wheresubstituent L is attached to the xanthene C4 carbon. In structures(Y-23a), (Y-24a) and (Y-25a), the C4 carbon in the illustrated tautomeris bridged to the xanthene nitrogen. Those of skill in the art willrecognize that due to tautomerism, the carbons labeled C4 and C5 (in thedefinition of rhodamine-type parent xanthene ring) are essentiallyequivalent. Thus, in the illustrated structures of compounds (Y-23a),(Y-24a) and (Y-25a), substituent L is bonded to the C5 carbon.Particularly preferred compounds according to this aspect of theinvention are those compounds in which:

[0210] Z is selected from the group consisting of (C₁-C₁₂) alkyl,(C₁-C₁₂) alkanyl, (C5-C₁₀) aryl, phenyl, naphthyl, naphth-1-yl,naphth-2-yl, pyridyl and purinyl; and/or

[0211] L is selected from the group consisting of (C₁-C₆) alkyldiyl,(C₁-C₆) alkano, (C₅-C₂₀) aryldiyl, phenyldiyl, phena-1,4-diyl,naphthyldiyl, naphtha-2,6-diyl, naphtha-2,7-diyl, (C₆-C₂₆) arylalkyldiyl—(CH₂)_(i)-φ-, —CH₂)_(i)-ψ-, —CH₂)_(i)—NHR″—C(O)-φ- and—CH₂)_(i)—NHR″—C(O)-ψ-, where each i is independently an integer from 1to 6, R″ is hydrogen or (C₁-C₆) alkyl φ is (C₅-C₂₀) aryldiyl, phenyldiylor phena-1,4-diyl and φ is naphthyldiyl, naphtha-2,6-diyl ornaphtha-2,7-diyl; and/or

[0212] R_(x) is any electrophilic or nucleophilic group.

[0213] In yet another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ib) in which Yis selected from the group consisting of (Y-1), (Y-2), (Y-3), (Y-4),where substituent L is attached to R^(3′), or one of the following groupof compounds, where R⁹ is as previously defined for structural formula(Y-1) and the dashed line at the nitrogen atom indicates the point ofattachment of substituent L:

[0214] In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formula (Ib) in which:

[0215] Y is selected from the group consisting of (Y-1), (Y-2), (Y-3),(Y-4), (Y-20b), (Y-21b), (Y-22b), (Y-23b), (Y-24b), (Y-25b), (Y-31b),(Y-34b), (Y-35b), (Y-36b), (Y-39b), (Y-42b), (Y-43b), and (Y-46b); and

[0216] Z is selected from the group consisting of (C₁-C₁₂) alkyl,(C₁-C₁₂) alkanyl, (C₅-C₁₀) aryl, phenyl, naphthyl, naphth-1-yl,naphth-2-yl, pyridyl and purinyl, where in structures (Y-1) through(Y-4) L is attached to R³.

[0217] L is selected from the group consisting of (C₁-C₆) alkyldiyl,(C₁-C₆) alkano, (C₅-C₂₀) aryldiyl, phenyldiyl, phena-1,4-diyl,naphthyldiyl, naphtha-2,6-diyl, naphtha-2,7-diyl, (C₆-C₂₆) arylalkyldiyl—(CH₂)_(i)-φ-, —(CH₂)_(i)-ψ-, —(CH₂)_(i)—NR″—C(O)-φ- and—(CH₂)_(i)—NR″—C(O)-ψ-, where each i is independently an integer from 1to 6, R″ is hydrogen or (C₁-C₆) alkyl, φ is (C₅-C₂₀) aryldiyl,phenyldiyl or phena-1,4-diyl and ψ is naphthyldiyl, naphtha-2,6-diyl ornaphtha-2,7-diyl, and where in structures (Y-1) through (Y-4), L isattached to R^(3′).

[0218] R_(x) is any electrophilic or nucleophilic group.

[0219] In still another preferred embodiment, the rhodamine dyes of theinvention are compounds according to structural formulae (I), (Ia) and(Ib), including any of their respective previously-described preferredembodiments, in which E is a carboxyl or a salt thereof.

[0220] In the rhodamine dyes of the invention, aside from substituent E,the exact positions of the various substituents substituting the newbottom ring, i.e., the Pyr⁺ and —S—Z¹—L—R_(x) substituents illustratedin structural formula (Ia) and the Pyr⁺ and —S—Z substituentsillustrated in structural formula (Ib) in the dyes of the presentinvention may be unknown. However, it has been confirmed by nuclearmagnetic resonance (NMR) that the bottom ring contains one, two, orthree Pyr⁺ substituents. Based upon the starting materials used tosynthesize the rhodamine dyes of the invention and the nature of thesynthesis reactions (discussed in more detail in Section 5.3.2, infra),it is believed that the various rhodamine dyes according to structuralformula (I) have the regiochemistry depicted in structural formula (Ic)(illustrated with Pyr⁺=4-(dimethylamino)pyridinium and E=—CO₂H):

[0221] It will be understood, however, that the Applicants do not intendto be bound by any particular structural representation, and that theinvention is intended to encompass the rhodamine dyes that are obtainedby the synthetic methods described herein, regardless of their specificregiochemistry.

[0222] In addition to the above-described structural isomerism about thenew bottom ring, those of skill in the art will appreciate that many ofthe compounds encompassed by formulae (I), (Ia) and/or (Ib) as describedherein, as well as many of the energy-transfer dye pairs and otherconjugates described infra, may exhibit the phenomena of tautomerism,conformational isomerism, geometric isomerism and/or stereoisomenism. Asthe formulae drawings within this specification can represent only oneof the possible tautomeric, conformational isomeric, enantiomeric orgeometric isomeric forms, it should be understood that the inventionencompasses any tautomeric, conformnational isomeric, enantiomericand/or geometric isomeric forms of the compounds having one or more ofthe utilities described herein.

[0223] Moreover, as the compounds of the invention may bear one or morepositive charges or negative charges, depending upon their physicalstate, they may be in the form of a salts or may have counterionsassociated therewith. The identity(ies) of any associated counterions istypically dictated by the synthesis and/or isolation methods by whichthe compounds are obtained. Typical counterions include, but are notlimited to, halides, acetate, trifluoroacetate, etc. and mixturesthereof. It will be understood that the identity(ies) of any associatedcounterions is not a critical feature of the invention, and that theinvention encompasses any type of associated counterion. Moreover, asthe compounds can exists in a variety of different forms, the inventionis intended to encompass not only forms that are in association withcounterions (e.g., dry salts), but also forms that are not inassociation with counterions (e.g., aqueous solutions).

[0224] 5.3.2 Methods of Synthesis

[0225] The rhodamine dyes of the invention, and in particular rhodaminedyes according to structural formula (Ia) in which each Pyr⁺ is4-(dimethylamino)pyridinium can be readily synthesized fromcorresponding tetrafluoro-rhodamine starting materials according toScheme I, below. Although a specific regiochemistry is depicted inScheme I, it will be understood that the structural representations areillustrative only. In Scheme I, Y, S, L, Z¹ and R_(x) are as previouslydefined for structural formula (Ia). E is illustratively a carboxyl, butcould be a sulfonate.

[0226] Referring to Scheme I, 4-(dimethyl)aminopyridine is added to asolution of tetrafluoro-rhodamine 100. The reaction is followed bythin-layer chromatography (TLC). The compound will depend upon theidentity of rhodamine-type parent xanthene ring Y. Once the reaction hasgone to completion, thiol 108 is added to the mixture and the reactionmonitored with TLC. Dye 110 is purified by reverse phase chromatographyor other standard methods. Compounds in which Pyr⁺ is other than4-(dimethylamino)pyridinium are prepared in an analogous manner from theappropriate aminopyridine starting material. Compounds havingnon-identical Pyr⁺ groups may be prepared using a mixture of theappropriate aminopyridines.

[0227] Compounds according to structural formula (Ib) are synthesized ina similar manner from the appropriate tetrafluoro-rhodamine derivative101 (illustrated below) using thiol Z—SH (106), where Z is as defined instructural formula (Ib), to displace a DMAP⁺ group:

[0228] In derivative 101, Y, L and are as previously defined forstructural formula (Ib). When linking moiety —L—R_(x) is attached at axanthene nitrogen, tetrafluoro-rhodamine 101 is prepared as illustratedin Scheme I. When linking moiety —L—R_(x) is attached at the xanthene C4position, tetrafluoro-rhodamine 101 is prepared from a substitutedmonomer 118 (R⁴=CH₂X or L—R_(X)). The full range of rhodamine dyesdescribed herein can be synthesized by routine modification of thesemethods. Where necessary, any reactive functionalities on linker L, e.g.reactive functional group R_(X) can be protected using known groups andmethods (see, eg., Greene & Wuts, Protective Groups in OrganicChemistry, 1991, 2^(nd) Edition, John Wiley & Sons, NY). Dyes in whichR_(X) is a carboxylic acid or a salt thereof are particularlyadvantageous, as the carboxyl group does not require protection duringsynthesis.

[0229] The appropriate tetrafluaoro-rhodamine starting materials can beproduced in the usual manner for the synthesis of rhodamines bycondensing 1 mol of an appropriate 3-aminophenol derivative with 1 molof tetrafluorophthalic anhydride 114 (Aldrich Chemical Co, St. Louis,Mo.) according to known techniques (see, e.g., U.S. Pat. No. 5,750,409;Römpps' Chemie Lexicon, 8^(h) Edition, pp. 3580). The appropriate3-aminophenol starting materials are either commercially available orcan be readily obtained using standard synthesis methods. Arepresentative synthesis for preparing tetrafluororhodamine startingmaterial 100 in which rhodamine-type parent xanthene ring Y is acompound according to structural formula (Y-1) (compound 120) isoutlined in Scheme II.

[0230] In Scheme II, the various R^(n) substituents are as previouslydefined for structural formula (Y-1). According to Scheme II, 1 mol of3-aminophenol derivative 112 is refluxed with 1 mol oftetrafluorophthalic anhydride 114 (Aldrich) to yield intermediate 116.Intermediate 116 is refluxed with 3-aminophenol derivative 118 to yieldtetrafluoro-rhodamine 120. Depending upon the identity of rhodamine-typeparent xanthene ring Y, the tetrafluoro-rhodamine 120 is isolated byreverse phase or normal phase column chromatography.

[0231] An analogous method for synthesizing tetrafluoro-rhodamine 100 inwhich rhodamine-type parent xanthene ring Y is a compound according tostructural formula (Y-2) is illustrated in Scheme III:

[0232] In Scheme Im, the various R^(n) substituents are as previouslydefined for structural formula (Y-2). Dyes according to structuralformula (Ia) in which the rhodamine-type parent xanthene ring is acompound according to structural formula (Y-3) or (Y-4) are prepared inan analogous manner from the appropriate starting materials.

[0233] Methods of preparing tetrafluoro-rhodamine derivative 101 inwhich Y is a compound according to structural formula (Y-1) or (Y-2) inwhich the linking moiety is attached to the xanthene nitrogen (ie., atposition R³) are illustrated in Schemes IIb and IIIb, respectively:

[0234] The various aminophenol starting materials are eithercommercially available or can be prepared using routine methods.Exemplary syntheses are provided in the Examples section. The full scopeof the rhodamine compounds described herein can be readily synthesizedby routine modification of any of these methods.

[0235] 5.4 Energy Transfer Dye Pairs

[0236] In another aspect, the present invention provides energy-transferdye pairs incorporating the new rhodamine dyes of the invention.Generally, the energy-transfer dye pairs of the present inventioncomprises three main elements: (i) a donor dye (DD) which absorbs lightat a first wavelength and emits excitation energy in response; (ii) anacceptor dye (AD) which is capable of absorbing the excitation energyemitted by the donor dye and emitting light at a second wavelength inresponse; and (iii) a linkage linking the donor dye to the acceptor dye,the linkage being effective to facilitate efficient energy-transferbetween the donor and acceptor dyes. In the energy-transfer dye pairs ofthe invention, at least one of the donor or acceptor dyes, typically theacceptor dye, is a new rhodamine dye of the invention. The donor andacceptor dyes can be linked together in a variety of differentconfigurations, depending upon the identities of the dyes. A thoroughdiscussion of the various structures, synthesis and use of certainenergy-transfer dye pairs which may aid an understanding of theenergy-transfer dye pairs of the invention is provided in U.S. Pat. No.5,800,996; U.S. Pat. No. 5,863,727; and U.S. Pat. No. 5,654,419, thedisclosures of which are incorporated herein by reference in theirentireties.

[0237] The energy-transfer dye pairs of the invention are typicallyobtained as illustrated in Scheme V:

[0238] In Scheme V, a dye 140 which includes an optional linker L″ and acomplementary functional group F_(x) is condensed with a rhodamine dyeof the invention according to structural formula (Ia) or (Ib) to yieldenergy-transfer dye pairs according to structural formulae (IIa) and(IIb), respectfully, where for example three pyridinium rings arepresent.

[0239] During the condensation, reactive group R and complementaryfunctional group F_(x) react to form covalent linkage R⁴¹. Thus, it willbe recognized by those of skill in the art that reactive group R_(x) andcomplementary functional group F_(x) can each constitute respectivemembers of the various pairs of complementary groups described inSection 5.3.1, such as various pairs of complementary electrophiles andnucleophiles. Preferably, one of R_(x) or F_(x) is an amine, thiol orhydroxyl group, most preferably an amine group, and the other one ofR_(x) or F_(x) is a group capable of reacting with an amine, thiol orhydroxyl, most preferably a carboxylic acid or a salt, ester oractivated ester thereof Thus, a particularly preferred covalent linkageR⁴′ is an amide of the formula —C(O)NR⁴⁵—, where R⁴⁵ is hydrogen or(C₁-C₆) alkyl.

[0240] In the energy-transfer dye pairs according to structural formulae(IIa) and (IIb), DD/AD represents either a donor dye or an acceptor dye.Whether DD/AD constitutes a donor or acceptor will depend on therespective excitation and emission properties of DD/AD and Y. Typically,DD/AD is a dye belonging to the xanthene (including fluoresceine andrhodamine dyes), cyanine, phthalocyanine or squaraine classes of dyes.Alternatively, DD/AD can be a dye of the invention, preferably a dyeaccording to structural formula (Ia) or (Ib). The only requirement isthat dye DD/AD either be capable of absorbing excitation energy emittedfrom Y or be capable of emitting excitation energy absorbable by Y.Preferably, in addition to complementary functional group F_(x), DD/ADincludes a linking moiety or linking group as previously described forconjugating the energy-transfer dye pairs of the invention to othercompounds or substances. L″ represents a bond or a linker analogous tothe previously described linkers L that, when included in anenergy-transfer dye pair according to structural formula (IIa) or (IIb),facilitates efficient energy transfer between the acceptor and donorchromophores. The identity of L″ and its point of attachment to DD/ADwill depend, in part, upon the identity of DD/AD. Structures of thesevarious classes of AD/DD dyes, as well as suitable linkers L″ and pointsof attachment to these various classes of AD/DD dyes are described inU.S. Pat. No. 5,863,727, the disclosure of which is incorporated hereinby reference. When DD/AD is a cyanine dye, L″ is preferably an amide,and is attached to the quaternary nitrogen . When DD/AD is aphthalocyanine dye, L″ is preferably a sulfonamide, and is attached tothe aromatic carbon skeleton. When DD/AD is a squaraine dye, L″ ispreferably an amide, and is attached to the quaternary nitrogen. WhenDD/AD is a rhodamine dye, UL is preferably an amide, and is attached toC4 or R⁹. When DD/AD is a fluorescein dye, L″ is preferably an amide,and is attached to C4 or R⁹.

[0241] Most preferably, DD/AD is a donor dye which emits excitationenergy absorbable by Y. Preferred donor dyes are xanthene dyes,especially those which include a linking moiety or linking group ontheir bottom rings. Preferred amongst the xanthene dyes are thefluorescein dyes. Fluorescein dyes suitable for use as donor dyesinclude, but are not limited to, the 4,7-dichlorofluoresceins describedin U.S. Pat. No. 5,188,934, the extended fluoresceins described in U.S.Pat. No. 5,775,409, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein(6-FAM), 5-(and 6)-carboxyfluorescein (5,6-FAM), NAN, Cl-FLAN and TET.Linker L″ is typically attached to the xanthene C4 position of thesepreferred fluorescein donor dye. The actual choice of donor dye willdepend upon the emission and excitation properties of DD/AD and Y, andwill be apparent to those of skill in the art. In preferred fluoresceindonor dyes that include a 5-, 6- or 5-(and 6)-carboxyl, the carboxylgroup (or an activated ester thereof) can be used to covalentlyconjugate the energy-transfer dye pairs of the invention to othercompounds or substances.

[0242] The energy-transfer dyes of the invention are more fullydescribed below with reference to various preferred embodiments.Referring to Scheme V, one group of preferred energy-transfer dye pairsaccording to structural formulae (IIa) and (IIb) are obtained when thecompounds according to structural formulae (Ia) and (Ib) are any oftheir previously-described preferred embodiments and compound 140 hasthe structure:

[0243] including any counter ions, wherein:

[0244] R³³ is hydrogen or

[0245] R³⁴, when taken alone, is hydrogen, or when taken together withR³⁵ is benzo;

[0246] R³⁵, when taken alone, is hydrogen, fluoro, chloro, hydroxyl orcarboxyl, or when taken together with R³⁴ is benzo;

[0247] R³⁶ is hydrogen, fluoro or chloro;

[0248] R³⁷, when taken alone, is hydrogen, fluoro, chloro, hydroxyl orcarboxyl, or when taken together with R³⁸ is benzo;

[0249] R³⁸, when taken alone, is hydrogen, or when taken together withR³⁷ is benzo;

[0250] R³⁹ is hydrogen or chloro;

[0251] R⁴⁰ is hydrogen or chloro; and

[0252] R⁵⁰ is carboxyl, or a salt, ester or activated ester thereofPreferred compound 140 can be the pure 5-isomer, the pure 6-isomer or amixture of 5-(and 6)-isomers. Particularly preferred compounds 140 arethose in which R³⁴, R³⁵, R³⁶, R³⁷, R³⁸, R³⁹ and R⁴⁰ are each hydrogen.

[0253] In another preferred embodiment, the energy-transfer dye pairs ofthe invention are compounds according to structural formula (IIa) inwhich:

[0254] Y is a compound according to structural formula (Y-1),(Y-2),(Y-3), (Y-4), (Y-20a), (Y-21a), (Y-22a), (Y-23a), (Y-24a), (Y-25a),(Y-31a), (Y-34a), (Y-35a), (Y-36a), (Y-39a), (Y-41a), (Y-42a), (Y-43a),(Y-44a), (Y-45a), or (Y-46a);

[0255] DD/AD is a fluorescein dye having a 5- or 6-carboxyl group or asalt, ester or activated ester thereof, particularly a fluorescein dyeselected from the group consisting of 5-carboxyfluorescein (5-FAM),6-carboxyfluorescein (6-FAM), 5-(and-6)-carboxyfluorescein, NAN, Cl-FLANand TET;

[0256] L is a bond, (C₁-C₆) alkyldiyl or (C₁-C₃) alkano, preferably abond;

[0257] Z¹ is (C₁-C₆)alkyldiyl, (C₅-C₁₄)aryl or heteroaryl;

[0258] R⁴¹ is —C(O)—R⁴²—, where R⁴² is O, S or NH and is bonded to L″;and/or

[0259] L″ is —R⁴³—Z³—C(O)—R⁴⁴—R⁴⁵—, wherein R⁴³ is (C₁-C₆) alkyldiyl,preferably (C₁-C₃) alkano, and is bonded to R⁴²; Z³ is 5-6 memberedcyclic alkenyldiyl or heteroalkenyldiyl, (C₅-C₁₄) aryldiyl orheteroaryldiyl; R⁴⁴ is O, S or NH; and R⁴⁵ is (C₁-C₆) alkyldiyl,preferably (C₁-C₃) alkano, and is bonded to the xanthene C4 carbon ofDD/AD.

[0260] Particularly preferred amongst the above-describedenergy-transfer dye pairs are those compounds in which:

[0261] L is a bond;

[0262] Z¹ and Z³ are each independently selected from the groupconsisting of phenyldiyl, phena-1,4-diyl, naphthyldiyl, naphtha-2,6-diyland naphtha-3,6-diyl;

[0263] R⁴² is NH;

[0264] R⁴³ is methano;

[0265] R:′ is NH; and/or

[0266] R⁴⁵ is methano.

[0267] In another preferred embodiment, the energy-transfer dye pairs ofthe invention are compounds according to structural formula (IIb) inwhich:

[0268] E is carboxyl or a salt thereof,

[0269] Y is a compound according to structural formula (Y-1), (Y-2),(Y-3) or (Y-4), where L is attached at position R^(3′), or is a compoundaccording to structural formula (Y-20b), (Y-21b), (Y-22b), (Y-23b),(Y-24b), (Y-25b), (Y-31b), (Y-34b), (Y-35b), (Y-36b), (Y-39b), (Y-41b),(Y-42b), (Y-43b), or (Y-46b);

[0270] DD/AD is a fluorescein dye having a 5- or 6-carboxyl group or asalt, an ester or an activated ester thereof, particularly a fluoresceindye selected from the group consisting of 5-carboxyfluorescein (5-FAM),6-carboxyfluorescein (6-FAM), 5-(and-6)-carboxyfluorescein, NAN, Cl-FLANand TET;

[0271] Z is (C₁-C₆) alkyl, (C₁-C₆) alkanyl, (C₅-C₁₄) aryl or heteroaryl;

[0272] L is —CH₂-φ- or —CH₂-ψ-, where φ is phenyldiyl and ψ isnaphthyldiyl and the methylene is attached to Y;

[0273] R⁴¹ is —C(O)-R⁴²—, where R⁴² is O, S or NH; and/or

[0274] L″ is —R⁴³—Z³—(O)—R⁴⁴—R⁴⁵—, wherein R⁴³ is (C₁-C₆) alkyldiyl,preferably (C₁-C₃) alkano, and is bonded to R⁴²; Z³ is 5-6 memberedcyclic alkenyldiyl or heteroalkenyldiyl, (C₅-C₁₄) aryldiyl orheteroaryldiyl; R⁴⁴ is O, S or NH; and R⁴⁵ is (C₁-C₆) alkyldiyl,preferably (C₁-C₃) alkano, and is bonded to the xanthene C4 carbon ofDD/AD.

[0275] Particularly preferred amongst the above-describedenergy-transfer dye pairs are those compounds in which:

[0276] L is methano;

[0277] Z is selected from the group consisting of phenyl, naphthyl,naphth-1-yl and naphth-2-yl;

[0278] Z³ is selected from the group consisting of phenyldiyl,phen-1,4-diyl, naphthyldiyl, naphth-2,6-diyl and naphth-3,6-diyl;

[0279] R⁴² is NH;

[0280] R⁴³ is methano;

[0281] R:′ is NH; and/or

[0282] R⁴⁵ is methano.

[0283] 5.4.1 Synthesis of the Energy-Transfer Dye Pairs

[0284] Methods for synthesizing the various energy-transfer dyes of theinvention are illustrated in Scheme V, supra. Conditions for carryingout the reactions are described in U.S. Pat. No. 5,863,727, includingCompound 140. Syntheses of exemplary energy-transfer dye pairs aredescribed in the Examples section. All of the energy-transfer dye pairsof the invention can be obtained by routine modification of any of thesemethods.

[0285] 5.5 Conjugates Incorporating Dyes and Energy-Transfer Dye Pairs

[0286] In another aspect, the present invention comprises moleculesand/or substances or conjugated with the rhodamine dyes and/orenergy-transfer dye pairs of the invention. The conjugates can comprisevirtually any molecule or substance to which the dyes or energy-transferdye pairs of the invention can be conjugated, including by way ofexample and not limitation, peptides, polypeptides, proteins,saccharides, polysaccharides, nucleosides, nucleotides, polynucleotides,lipids, solid synthesis supports, organic and inorganic polymers, andcombinations and assemblages thereof, such as chromosomes, nuclei,living cells (e.g., bacteria or other microorganisms, mammalian cells,tissues, etc.), and the like. The dyes or energy-transfer dye pairs areconjugated with the reagent via a linking moiety by a variety of means,including hydrophobic attraction, ionic attraction, covalent attachmentor with the aid of pairs of specific binding molecules, as previouslydescribed. Preferably, the dyes are conjugated via covalent attachment.

[0287] Conjugation typically results from mixing a dye or dye pairincluding an optional linking moiety with and the molecule or substanceto be conjugated in a suitable solvent in which both are soluble, usingmethods well-known in the art, followed by separation of the conjugatefrom any unconjugated starting materials or unwanted by-products. Thedye or dye pair conjugate can be stored dry or in solution for lateruse.

[0288] 5.5.1 Nucleoside/Tide Conjugates

[0289] A preferred class of conjugates include nucleosides/tides andnucleoside/tide analogs that are labeled with the rhodamine dyes orenergy-transfer dye pairs of the invention. Such labelednucleosides/tides are particularly useful for labeling polynucleotidesformed by enzymatic synthesis, e.g., labeled nucleotide triphosphatesused in the context of template-directed primer extension, PCRamplification, Sanger-type polynucleotide sequencing, and/ornick-translation reactions. Labeled nucleoside/tides and/ornucleoside/tide analog conjugates are generally obtained by condensing anucleoside/tide or nucleoside/tide analog (NUC) modified to contain alinking moiety (—L′—F_(x)) with a rhodamine dye according to structuralformula (Ia) or (Ib) to yield a dye-labeled nucleoside/tide ornucleoside/tide analog according to structural formula (IIIa) or (IIIb),respectively. Energy-transfer dye pair-labeled nucleosides/tides and/ornucleoside/tide analogs are obtained in an similar manner withenergy-transfer dye compounds according to structural formulae (IIa) and(IIb) in which the DD/AD dye includes an optional linking moiety of theformula —L³—R_(x), where L³ is a bond or linker similar topreviously-described linker L (compounds (IIa.1) and (IIb.1),respectively). These reactions are illustrated in Schemes VIa and Vib:

[0290] Referring to Schemes VIa and VIb, where for example threepyridinium rings are present and reactive functional group R_(x) on dyesor energy-transfer dye pairs according to structural formulae (Ia),(Ib), (IIa.1) and/or (IIb.1) and complementary functional group F_(x) oncompound 144 react to form covalent linkage R⁴⁶. Covalent linkage R⁴⁶ isanalogous to the previously-described covalent linkage R⁴¹. Preferredembodiments of R⁴⁶ are the same as the preferred embodiments previouslydescribed for R⁴¹.

[0291] Complementary functional group F_(x) is attached to NUC vialinker L′. Complementary functional group F_(x) may be attached directlyto NUC, in which case L′ represents a bond, or it may be spaced awayfrom NUC by one or more intervening atoms, in which case L′ represents alinker. Any of the linkers L or L′ previously described in connectionwith the rhodamine dye compounds per se and/or the energy-transfer dyepairs per se can be used for linker L′.

[0292] Complementary functional group F_(x) may be attached to NUC at avariety of different positions, e.g., the nucleobase, the sugar and/orthe phosphate ester moiety. Nucleosides/tides and nucleoside/tideanalogs that are appropriately modified at these various positions suchthat they can be conjugated with dyes and/or dye pairs according to theinvention are known in the art. Preferably, complementary group F, isattached to the nucleobase. When the nucleobase is a 7-deazapurine, U isusually attached to the C7 position of the nucleobase. When thenucleobase is a pyrimidine, L′ is usually attached to the C-5 positionof the nucleobase. When the nucleobase is a purine, L′ is usuallyattached to the C-8 position of the nucleobase. Linkers L′ useful forcovalently conjugating the dyes of the invention to the nucleobase ofNUC are described in U.S. Pat. No. 5,821,356, U.S. Pat. No. 5,770,716and U.S. application Ser. No. 08/833,854 filed Apr. 10, 1997, thedisclosures of which are incorporated herein by reference.

[0293] Preferred linkers L′ for covalently conjugating the dyes of theinvention to the nucleobase of NUC include (C₂-C₂₀) alkylenos,heteroalkyldiyls and heteroalkylenos, especially (C₂-C₂₀) alkynos,(C₂-C₂₀) alkenos, heteroalkynos and heteroalkenos. A particularlypreferred linker L′ is —C≡C—CH₂—, where the terminal sp carbon iscovalently attached to the nucleobase of NUC and the terminal methylene(sp3) carbon is covalently attached to R⁴⁶ in the compounds ofstructural formulae (IIIa), (IIIb), (IVa) and (IVb), or to F_(x) ofcompound 144.

[0294] Additional preferred linkers L′ for covalently conjugating therhodamine dyes or energy-transfer dye pairs of the invention to thenucleobase of NUC include propargylethoxy groups according to structuralformula —C≡C—CH₂—O—CH₂—CH₂—NR⁴⁷—R⁴⁸—, wherein R⁴⁷ is hydrogen or (C₁-C₆)alkyl and R⁴⁸ is selected from the group consisting of —C(O)—(CH₂)_(r)—,—C(O)—CHR⁴⁹—, —C(O)—C≡C—CH₂— and C(O)-φ-(CH₂)_(r)—, where each r isindependently an integer from 1 to 5 and 4 represents a C₆ aryldiyl orheteroaryldiyl, preferably phena-1,4-diyl

[0295] and R⁴⁹ is hydrogen, (C₁-C₆) alkyl or an amino acid side chain(including side chains of both gene-encoded and non-encoded aminoacids). With these linkers L′, the terminal sp carbon is attached to thenucleobase of NUC and the other terminal group is attached to R⁴⁶ in thecompounds of structural formulae (IIIa), (IIIb), (IVa) and (IVb), or toF_(x) of compound 144.

[0296] In a preferred embodiment, the labeled nucleosides/tide and/ornucleoside/tide analogs according to structural formulae (IIIa), (IIIb),(IVa) and (IVb) are labeled enzymatically-incorporatable nucleotides,labeled enzymatically extendable nucleotides or labeled terminators.

[0297] In another preferred embodiment, the labeled nucleosides/tidesand nucleoside/tide analogs are those obtained from Schemes (VIa) and(VIb) in which the compounds according to structural formulae (Ia), (Ib)are any of their respective previously-defined preferred embodiments,compounds (IIa.1) and (IIb.1) are any of the previously definedpreferred embodiments of compounds (IIa) and (IIb), respectively, andcompound 144 is a compound according to structural formula (V):

[0298] wherein:

[0299] B is a nucleobase;

[0300] F_(x) is a complementary functional group as previouslydescribed;

[0301] L′ is one of the preferred linkers described above;

[0302] R₇₀ and R₇₁, when taken alone, are each independently selectedfrom the group consisting of hydrogen, hydroxyl and a moiety whichblocks polymerase-mediated template-directed nucleic acid synthesis, orwhen taken together form a bond such that the illustrated sugar is2′,3′-didehydroribose; and

[0303] R₇₂ is selected from the group consisting of hydroxyl, aphosphate ester having the formula

[0304] where a is an integer from 0 to 2, and a phosphate ester analog.Typically, F_(x) is an amino group of the formula —NHR⁵¹, where R⁵¹ shydrogen or (C₁-C₆)alkyl, but can be any nucleophilic or electrophilicgroups.

[0305] In a preferred embodiment of structural formula (V), B is anormal nucleobase or a common analog thereof, a 7-deazapurine, 8-aza,7-deazapurine, a purine or a pyrimidine. In a particularly preferredembodiment, B is a nucleobase selected from the group consisting of7-deaza-adenine, cytosine, 7-deaza-guanine, thymine and uracil. When thepreferred nucleobase B is a purine or a 7-deaza-purine, the pentosemoiety is attached to the N⁹-position of the nucleobase, and when thepreferred B is a pyrimidine, the pentose moiety is attached to theN¹-position of the nucleobase. Linker L′ is attached to nucleobase B aspreviously described.

[0306] In structural formula (V), when both R₇₀ and R₇₁ are hydroxyl,the resultant compounds produced in Schemes VIa and VIb are labeledribonucleoside/tides. When R₇₀ is hydrogen and R₇₁ is hydroxyl, theresultant compounds are labeled 2′-deoxyribonucleoside/tides. When R₇₀and R₇₁ are each hydrogen, the resultant compounds are2′,3′-dideoxyribonucleoside/tides Labeled2′,3′-dideoxyribnucleoside-5′-triphosphates (ddNTPs) find particular useas terminators in Sanger-type DNA sequencing methods utilizingfluorescent detection. Labeled 2′-deoxyribonucleoside-5′-triphosphates(dNTPs) find particular use as means for labeling DNA polymeraseextension products, e.g., in the polymerase chain reaction ornick-translation. Labeled ribonucleoside-5′-triphosphates (NTPs) findparticular use as means for labeling RNA polymecrase extension products.

[0307] Referring to Schemes (VIa) and (VIb), supra, the synthesis ofalkynylamino-derivatized compounds 144 useful for conjugating the dyesof the invention to nucleosides/tides and/or nucleoside/tide analogs istaught in EP 87305844.0 and Hobbs et al., 1989, J. Org. Chem. 54:3420.The corresponding nucleoside mono-, di- and triphosphates are obtainedby standard techniques (see, e.g., the methods described in U.S. Pat.No. 5,821,356, U.S. Pat. No. 5,770,716 and U.S. application Ser. No.08/833,854 filed Apr. 10, 1997, discussed supra). Methods forsynthesizing compound 144 in modified with propargylethoxyarrido linkersL′ can also be found in these patents and application.

[0308] Energy-transfer dye pairs can be conjugated to a nucleotide5-triphosphate 144 by linling through a nucleobase amino group to: (i)an activated ester of a energy-transfer dye pair, e.g. 230 NHS ester, or(ii) stepwise coupling to one dye 140, e.g. 4′-protected aminomethylfluorescein, then coupling the unprotected 4′-aminomethyl to the seconddye of the pair, e.g. 196 NHS ester.

[0309] Additional synthesis procedures suitable for use in synthesizingcompounds according to structural formulae (IIIa), (IIIb), (IVa) and(IVb) are described, for example, in Gibson et al., 1987, Nucl. AcidsRes. 15:6455-6467; Gebeyehu et al., 1987, Nucl. Acids Res. 15:4513-4535;Haralambidis et al., 1987, Nucl. Acids Res. 15:4856-4876; Nelson et al.,1986, Nucleosides and Nucleotides. 5(3):233-241; Bergstrom et al., 1989,J. Am. Chem. Soc. 111:374-375; U.S. Pat. No. 4,855,225, U.S. Pat. No.5,231,191 and U.S. Pat. No. 5,449,767, the disclosures of which areincorporated herein by reference. Any of these methods can be routinelyadapted or modified as necessary to synthesize the full range of labelednucleosides/tides and nucleoside/tide analogs described herein.

[0310] 5.5.1.1 Polynucleotide Conjugates

[0311] Yet another preferred class of conjugates of the presentinvention comprise polynucleotides and/or polynucleotide analogs labeledwith the rhodamine dyes or energy-transfer dye pairs of the invention.Such labeled polynucleotides and/or analogs are useful in a number ofimportant contexts, including as DNA sequencing primers, PCR primers,oligonucleotide hybridization probes, oligonucleotide ligation probes,and the like.

[0312] In one preferred embodiment, the labeled polynucleotides orpolynucleotide analogs of the present invention include multiple dyeslocated such that fluorescence energy-transfer takes place between adonor dye and an acceptor dye. Such multi-dye energy-transferpolynucleotides find application as spectrally-tunable sequencingprimers as described, for example, in Ju et al., 1995, Proc. Natl. Acad.Sci. USA 92:4347-4351, and as hybridization probes as described, forexample, in Lee et al., 1993, Nucl. Acids Res. 21:3761-3766.

[0313] Labeled polynucleotides and/or polynucleotide analogs aretypically synthesized enzymatically, e.g., using a DNA/RNA polymerase orligase (see, e.g., Stryer, 1981, Biochemistry, Chapter 24, W.H. Freemanand Company) and a labeled enzymatically-incorporatable nucleotide, aspreviously described. Alternatively, the labels may be introducedsubsequent to synthesis via conventional conjugation reactions, asdiscussed more thoroughly below.

[0314] Generally, if the labeled polynucleotide is made using enzymaticsynthesis, the following procedure may be used. A target DNApolynucleotide is denatured and an oligonucleotide primer is annealed tothe target DNA. A mixture of 2′-deoxyribonucleoside-5′-triphosphatescapable of supporting template-directed enzymatic extension of theprimed target (e.g., a mixture including dGTP, dATP, dCTP, and dTTP) isadded to the primed target. At least a fraction of the deoxynucleotidesare labeled with a rhodamine dye or energy-transfer dye pair of theinvention as described above. Next, a polymerase enzyme is added to themixture under conditions where the polymerase enzyme is active. Alabeled polynucleotide is formed by the incorporation of the labeleddeoxynucleotides during polymerase-mediated strand synthesis. In analternative enzymatic synthesis method, two primers are used instead ofone: one complementary to the plus (+) strand of the target and anothercomplementary to the minus (−) strand of the target, the polymerase is athermostable polymerase and the reaction temperature is cycled between adenaturation temperature and an extension temperature, therebyexponentially synthesizing a labeled complement to the target sequenceby PCR (see, e.g., PCR Protocols, 1990, Innis et al. Eds., AcademicPress).

[0315] Alternatively, the labeled polynucleotide or polynucleotideanalog is obtained via post-synthesis conjugation. According to thisembodiment, a polynucleotide or polynucleotide analog which includes acomplementary functional group F_(x), typically an amino group, iscondensed with a rhodamine dye according to structural formula (IIa) or(IIb) or an energy-transfer dye pair according to structural formula(IIIa) or (IIIb) under conditions wherein R_(x) and F_(x) react to forma covalent linkage. The labeled polynucleotides and/or polynucleotideanalogs are isolated using conventional means, such as alcoholprecipitation, gel electrophoresis, column chromatography, etc.

[0316] A variety of reagents for introducing amino groups, or othercomplementary functional groups such as thiols into enzymatically orchemically synthesized polynucleotides and polynucleotide analogs areknown in the art, as are appropriate condensation conditions. Forexample, methods for labeling polynucleotides at their 5′-terrinus aredescribed in Oligonucleotides and Analogs, 1991. Eckstein, Ed., Chapter8, MRL Press; Orgel et al., 1983, Nucl. Acids Res. 11(18):6513; and U.SPat. No. 5,118,800. Methods for labeling polynucleotides at thephosphate ester backbone are described in Oligonucleotides and Analogs,1991. Eckstein, Ed., Chapter 9, IRL Press. Methods for labelingpolynucleotides at their 3′-terminus are described in Nelson et al.,1992, Nucl. Acids Res. 20(23):6253-6259; U.S. Pat. No. 5,401,837; andU.S. Pat. No. 5,141,813. For a review of labeling procedures, the readeris referred to Haugland, In: Excited States of Biopolymers, 1983,Steiner. Ed., Plenum Press, NY. All of these disclosures areincorporated herein by reference.

[0317] 5.6 Methods Utilizing the Dyes and Reagents of the Invention

[0318] The rhodamine dyes, energy-transfer energy-transfer dye pairs andconjugates incorporating the dyes and energy-transfer dye pairs of thepresent invention are well suited to any method utilizing fluorescentdetection, particularly aqueous applications and methods requiring thesimultaneous detection of multiple spatially-overlapping analytes. Thevarious dyes and conjugates of the invention are particularly wellsuited for identifying classes of polynucleotides that have beensubjected to a biochemical separation procedure, such aselectrophoresis, or that have been distributed among locations in aspatially-addressable hybridization array.

[0319] The various dyes of the invention can be conjugated to peptides,proteins, antibodies, and antigens. Dye-antibody conjugates are usefulfor sandwich-type immunosorbent assays. Especially preferred arebead-based assays for detection of peptides, cells, and other cellularcomponents where the dyes of the invention are conjugated to antibodies.

[0320] In a preferred category of methods referred to herein as“fragment analysis” or “genetic analysis” methods, labeledpolynucleotide fragments are generated through template-directedenzymatic synthesis using labeled primers or nucleotides, e.g., byligation or polymerase-directed primer extension; the fragments aresubjected to a size-dependent separation process, e.g, electrophoresisor chromatography; and, the separated fragments are detected subsequentto the separation, e.g., by laser-induced fluorescence. In aparticularly preferred embodiment, multiple classes of polynucleotidesare separated simultaneously and the different classes are distinguishedby spectrally resolvable labels.

[0321] One such fragment analysis method known as amplified fragmentlength polymorphism detection (AmpFLP) is based on amplified fragmentlength polymorphisms, i.e., restriction fragment length polymorphismsthat are amplified by PCR. These amplified fragments of varying sizeserve as linked markers for following mutant genes through families. Thecloser the amplified fragment is to the mutant gene on the chromosome,the higher the linkage correlation. Because genes for many geneticdisorders have not been identified, these linkage markers serve to helpevaluate disease risk or paternity. In the AmpFLPs technique, thepolynucleotides may be labeled by using a labeled polynucleotide PCRprimer, or by utilizing labeled nucleotide triphosphates in the PCR.

[0322] In another such fragment analysis method known as nicktranslation, a reaction is used to replace unlabeled nucleotides in adouble-stranded (ds) DNA molecule with labeled nucleotides. Free3′-hydroxyl groups are created within the dsDNA by “nicks” caused bytreatment with deoxyribonuclease I (DNAase D). DNA polymerase I thencatalyzes the addition of a labeled nucleotide to the 3′-hydroxylterminus of the nick. At the same time, the 5′ to 3′-exonucleaseactivity of this enzyme eliminates the nucleotide at the 5′-phosphorylterminus of the nick. A new nucleotide with a free 3′-OH group isincorporated at the position of the original excised nucleotide, and thenick is shifted along by one nucleotide in the 3′ direction. This 3′shift will result in the sequential addition of new labeled nucleotidesinto the dsDNA. The nick-translated polynucleotide is then analyzed, forexample, by using a separation process such as electrophoresis.

[0323] Another exemplary fragment analysis method is based on variablenumbers of tandem repeats, or VNThs. VNTRs are regions ofdouble-stranded DNA that contain multiple adjacent copies of aparticular sequence, with the number of repeating units being variable.Examples of VNTR loci are pYNZ22, pMCT118, and Apo B. A subset of VNTRmethods are those methods based on the detection of microsatelliterepeats, or short tandem repeats (STRs), i.e., tandem repeats of DNAcharacterized by a short (2-4 bases) repeated sequence. One of the mostabundant interspersed repetitive DNA families in humans is the(dC-dA)n-dG-dT)n dinucleotide repeat family (also called the (CA)ndinucleotide repeat family). There are thought to be as many as 50,000to 100,000 (CA)n repeat regions in the human genome, typically with15-30 repeats per block. Many of these repeat regions are polymorphic inlength and can therefore serve as useful genetic markers. Preferably, inVNTR or STR methods, label is introduced into the polynucleotidefragments using a labeled PCR primer.

[0324] In a particularly preferred fragment analysis method, classesidentified in accordance with the invention are defined in terms ofterminal nucleotides so that a correspondence is established between thefour possible terminal bases and the members of a set of spectrallyresolvable dyes. Such sets are readily assembled from the dyes andenergy-transfer dye pairs of the invention by measuring emission andabsorption bandwidths with commercially available spectrophotometers.More preferably, the classes arise in the context of the chemical orchain termination methods of DNA sequencing, and most preferably theclasses arise in the context of the chain termination methods, i.e.,dideoxy DNA sequencing, or Sanger-type sequencing.

[0325] Sanger-type sequencing involves the synthesis of a DNA strand bya DNA polymerase in vitro using a single-stranded or double-stranded DNAtemplate whose sequence is to be determined. Synthesis is initiated at adefined site based on where an oligonucleotide primer anneals to thetemplate. The synthesis reaction is terminated by incorporation of aterminator that will not support continued DNA elongation. When properproportions of dNTPs and a single terminator complementary to A, G, C orT are used, enzyme-catalyzed primer extension will be terminated in afraction of the extension products at each site where the terminator isincorporated. if labeled primers or labeled terminators are used foreach reaction, the sequence information can be detected by fluorescenceafter separation of the resultant primer extension products byhigh-resolution electrophoresis. In the chain termination method, dyesor energy-transfer dye pairs of the invention can be attached to eitherthe sequencing primers or terminators. The dyes or energy-transfer dyepairs can be linked to a complementary functionality on the 5′-end ofthe primer, e.g. following the teaching in Fung et al, U.S. Pat. No.4,757,141; on the base of a primer; or on the base of a terminator, e.g.via the alkynylamino or other linking groups described above.Concentration ranges for the various enzymes, primers, dNTPs and labeledterminators are those commonly employed in the art.

[0326] In each of the above fragment analysis methods, labeled extensionproducts are preferably separated by electrophoretic procedures, e.g.Gel Electrophoresis of Nucleic Acids: A Practical Approach, 1981,Rickwood and Haames, Eds., IRL Press Limited, London; Osterman, 1984,Methods of Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag,Berlin; or U.S. Pat. Nos. 5,374,527, 5,624,800 and/or 5,552,028.Preferably, the type of electrophoretic matrix is crosslinked oruncrosslinked polyacrylamide having a concentration (weight to volume)of between about 2-20 weight percent. More preferably, thepolyacrylamide concentration is between about 4-8 percent. Preferably,in the context of DNA sequencing, the electrophoresis matrix includes adenaturing agent, e.g., urea, formamide, and the like. Detailedprocedures for constructing such matrices are given by Maniatis et al.,1980, “Fractionation of Low Molecular Weight DNA and RNA inPolyacrylamide Gels Containing 98% Formamide or 7 M Urea,” Methods inEnzymology 65:299-305; Maniatis et al., 1975, “Chain LengthDetermination of Small Double- and Single-Stranded DNA Molecules byPolyacrylamide Gel Electrophoresis,” Biochemistry 14:3787-3794; Maniatiset al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York, pgs. 179-185; and ABI PRISM™ 377 DNA SequencerUser's Manual, Rev. A, January 1995, Chapter 2 (p/n 903433, ThePerkin-Elmer Corporation, Foster City, Calif.). The optimalelectrophoresis conditions, e.g., polymer concentration, pH,temperature, and concentration of denaturing agent, employed in aparticular separation depend on many factors, including among others,the size range of the nucleic acids to be separated, their basecompositions, whether they are single stranded or double stranded, andthe nature of the classes for which information is sought byelectrophoresis. Accordingly, application of the invention may requirestandard preliminary testing to optimize conditions for particularseparations.

[0327] Subsequent to electrophoretic separation, the labeled extensionproducts are detected by measuring the fluorescence emission from thelabels. To perform such detection, the labeled products are illuminatedby standard means, e.g high intensity mercury vapor lamps, lasers, orthe like. The illumination wavelength will depend upon the spectralproperties of the particular label. Preferably, the illumination meansis a laser having an illumination beam at a wavelength between 400 and700 nm. More preferably, the illumination means is a laser lightgenerated by an argon ion laser, particularly the 488 and 514 nmemission lines of an argon ion laser, or the 532 emission line of aneodymium solid-state YAG laser or the 633 um emission line of ahelium-neon laser. Several argon ion lasers are available commerciallywhich lase simultaneously at these lines, e.g Cyonics, Ltd. (Sunnyvale,Calif ) Model 2001, or the like. The fluorescence is then detected by alight-sensitive detector, e.g., a photomultiplier tube, a chargedcoupled device, or the like. Suitable exemplary electrophoresisdetection systems are described elsewhere, e.g., U.S. Pat. Nos.5,543,026; 5,274,240; 4,879,012; 5,091,652 and 4,811,218.

[0328] In preferred embodiments, the primer is unlabeled and thesequencing reaction includes, in addition to the polymerase and mixtureof dNTPs, a mixture of four different terminators, one complementary toA, one complementary to G, one complementary to C and one complementaryto T. Each of the different terminators is labeled with a different,spectrally resolvable dye or energy-transfer dye pair. One of theterminators is labeled with a rhodamine dye or energy-transfer dye pairof the invention. As each of the labeled terminators fluoresces at adifferent wavelength, following separation based on size, the identityof the 3′-terminal nucleotide of each extension product is identified bythe wavelength (or color) of the label. In particularly preferredembodiments, each of the different spectrally resolvable labels can beexcited using a single light source. A set of such preferred labeledterminators is provided in the Examples section. Other sets will dependupon the excitation and emission spectral properties of the variouslabels are readily obtained by routine methods.

[0329] The invention having been described, the following examples areprovide to illustrate, and not limit, the invention.

6. EXAMPLE Synthesis of Rhodamine Dye 190

[0330] Rhodamine dye 190 was synthesized as illustrated in Schemes I andII, supra, from the appropriate aminophenol starting materials.

[0331] 6.1 Synthesis of 202

[0332] Amino-phenol compound 200 was synthesized as described in U.S.Pat. No. 5,750,409 by alkylation of 192 with ethyl iodide and sodiumbicarbonate in acetonitrile followed by demethylation with borontribromide in dichloromethane.

[0333] A solution of aminophenol 200 (4.8 g, 22 nunol) andtetrafluorophthalic anhychide 114 (4.9 g, 22 mmol; Aldrich) was refluxedin toluene (44 ml) for 3 hr. The solution was cooled to rt and theprecipitate collected to yield tetrafluoro ketone 202 (6.8 g, 70%/).

[0334] 6.2 Synthesis of 204

[0335] A suspension of the tert-butyl ester amine 194, preparedaccording to U.S. Pat. No. 5,688,808, (5.46 g, 20 mmol), methyl4-(bromomethyl)benzoate (8.3 g, 36.2 mmol), sodium iodide (3.0 g, 20mmol) and potassium carbonate (2.8 g, 20 mmol) was refluxed inacetonitrile (20 ml) for 1 hr. TLC (hexane/ethyl acetate 1/1) showed thereaction was complete. Ether was added to the cooled mixture and thesolution was decanted from the solids. The solution was washed withwater (2 portions of 50 ml), brine (50 ml) and dried over magnesiumsulfate. The crude product was purified by chromatography on silica gel(hexane/ethyl acetate 19:1) to give white crystals of the alkylated,tert-butyl ester product (4.3 g, 10 mmol, 50%/o). The tert-butyl esterwas dissolved in a solution of lithium hydroxide monohydrate (1.7 g, 40mmol) in water (10 ml) and methanol (50 ml), refluxed for 2 hr and thenevaporated to dryness under vacuum. The residue was extracted withdiethylether (100 ml) and washed with water and brine. The organic layerwas dried over magnesium sulfate, filtered and evaporated to give 204 asa tan foam. ¹H NMR (204, CD₂Cl₂) δ 8.03, 2H, d; 7.45, 2H, d; 6.96, 1H,d; 6.04, 1H, d; 5.65, 1H, s; 4.55, 1H, m; 1.38, 3H, s; 1.25, 6H, s.

[0336] 6.3 Synthesis of 206

[0337] Phosphorous oxytrichloride (2.8 ml, 30 mmol) was added to asuspension of tetrafluoro ketone 202 (4.4 g, 10 mmol) in chloroform (100ml). The suspension was stirred at rt for 30 min., aminophenol 204 (3.2g, 10 mmol) was added and the mixture was refluxed for 3 hr. Thesolution was cooled to rt and the reaction quenched with water (1 ml).The solvent was evaporated and dye 206 was purified by normal phasechromatography (DCM/4eOH/HOAc, 90:10:1). Dye 206 was further purified byC18 reverse phase chromatography (MeOH/0.1 M TEAA, 4:1) to afford a darkgreen solid (0.65 g, 9%, Abs. max 610 nm, Em. max 632 nm, H₂O).

[0338] 6.4 Svnthesis of Dye 190

[0339] Dimethylaminopyridine (85 mg) was added to tetrafluoro dye 206(30 mg) in 0.7 ml of dimethylformamide. After 20 hours at roomtemperature, TLC analysis (CH₂Cl₂:CH₃OH:CH₃CO₂H 80:20:16) showed thecomplete disappearance of starting 206 and the appearance of a new,lower Rf spot, the tetradimethylaminopyiidinium adduct Thiophenol (0.75ml) was added, and after another 8 hours, TLC showed partial conversionto a higher Rf spot. After concentration under vacuum, 190 was purifiedby reverse-phase TPLC (C₁₈) by a gradient of 30-50% acetonitrile in 0.1MTEAA. The fractions containing 190 were combined and evaporated to givea blue oil which was precipitated in either to provide pure dye 190 as ablue solid (30 mg, Abs. Max 628 nm, Em. max 650 nm, H₂O).

[0340] The succinimidyl (NHS) ester of 190 was prepared from a solutionof 190 (5 mg) in 100 μl dimethylformamide (DMF). Succinimidyltetramethyluronium tetrafluoroborate (20 mg) and diisopropylammie (10μl) were added. After 1 hour at room temperature, TLC analysis(C2-reverse phase plates, CH₃OH/0.1M TEAA 1:1) showed the disappearanceof starting 190 and a new, lower Rf spot. The NHS ester of 190 wasisolated by precipitation as ablue solid.

[0341] 7. EXAMPLE

Synthesis of Rhodamine Dye 196

[0342] Rhodamine dye 196 was synthesized in a manner analogous to dye190 from the corresponding tetrafluoro dye 220.

[0343] 7.1 Synthesis of Tetrafluoro 220

[0344] Phosphorous oxytrichloride (2.8 ml, 30 mmol) was added to asuspension of tetrafluoro ketone 202 (4.4 g, 10 mmol) in chloroform (100ml). The suspension was stirred at rt for 30 min., aminophenol 200 (3.2g, 10 mmol) was added and the mixture was refluxed for 3 hr. Thesolution was cooled to rt and the reaction quenched with water (1 ml).The solvent was evaporated and dye 220 was purified by normal phasechromatography (DCM/MeOH/HOAc, 90:10:1). Dye 220 was further purified byC₁₈ reverse phase chromatography (CH₃OH:0.1M TEAA, 4:1) to afford a darkgreen solid (0.65 g, 9%, Abs. max 613 nm, Em. max 643 mu, H₂O).

[0345] 7.2 Synthesis of Rhodamine Dye 196

[0346] To a solution of 220 (100 mg) in dimethylformamide (1.5 mL) wasadded 4-(dimethylamino)pyridine (150 mg). Thin-layer chromatography(TLC) on silica gel eluting with 80:20:16dichioromethane:methanol:acetic acid could distinguiish tetrafluoro 220(Rf=1) from the tetra-dimethylaminopyridinium adduct (Rf=0). After 40hr, analysis by thin-layer chromatography showed complete conversion tothe SymJAZ adduct.

[0347] To this solution, 4-carboxythiobenzene (50 mg) was added. After10 min, TLC analysis showed conversion to rhodamine dye 196 (Rf=0.1).Purification of 196 was accomplished on C18 silica gel with stepwiseelution with 20-70% methanol vs. 0.1 M triethylammonium acetate. The dye196 eluted between 30% and 50% methanol. The solvent was evaporated andthe residual blue oil was precipitated with ether to provide the dye 196as a blue solid (85 mg, Abs. max 636 nm, Em. max 661 mun, H₂O).

[0348] 7.3 Synthesis of Tetrafluoro 198

[0349] Amino-phenol compound 228 was synthesized as described in U.S.Pat. No. 5,750,409 -and cyclized with tetrafluorophthalic anhydride 114as in Example 6.1 supra to give 238 (lambda max 384 nm). The mixture of2.6 mmole 238 and 2.6 mmole 228 in 1.2 gm phosphorus oxytrichloride and10 ml acetonitrile was refluxed for 12 hours. The solvent was evaporatedunder vacuum, dissolved in several milliliters of dichloromethane andchromatographed on silica gel, eluting with dichloromethane andmethanol. The product fractions were combined, evaporated and furtherpurified by reverse-phase preparative HPLC and precipitation in 1% HClto give 35 mg 198 as a red solid. ¹H NMR (198, CD₃OD) δ 6.82, 211, s;6.59, 2H, s; 5.63,211, s; 1.9, 6H, br s; 1.4, 121, br s.

[0350] 7.4 Syntesis of Tetrafluoro 240

[0351] Tetrafluoro 240 was synthesized by the method and correspondingmethylated intermediates of Example 7.3. Mass spectroscopy 240: ExactMass=662.22 (Molecular Wt.=662.67) Ex. max 624 nm, Em. max 644 nm (8Murea).

8. EXAMPLE Synthesis of Rhodamine Dye 232

[0352]

[0353] Rhodamine dye 232 was synthesized as illustrated in Schemes (I)and (II), supra, from the appropriate aminophenol starting mnaterials.

[0354] 8.1 Synthesis of Triadcic Amine-Phenol 208

[0355] A suspension of the tert-butyl ester amine 194, preparedaccording to U.S. Pat. No. 5,688,808, (12.8 g, 47 mmol),1-bromo-3-chloropropane (29.3 g, 187 mmol), sodium iodide (56.4 g, 376mmol) and sodium bicarbonate (7.9 g, 94 mmol) was refluxed inacetonitrile (150 ml) for 18 hr. The mixture was cooled to roomtemperature, filtered by suction filtration and evaporated to leave aresidue. The filter cake was washed with hexane (300 ml) and thefiltrate was combined with the residue and washed with water (2 portionsof 50 ml), brine (50 ml) and dried over magnesium sulfate. The crudecyclized product was purified by chromatography on silica gel(hexane/ethyl acetate 20:1) to leave the intermediate tricyclic pivalateester as a pale yellow oil (9.5 g, 30 mmol, 64%). The cyclized ester wasdissolved in a solution of lithium hydroxide monohydrate (2.6 g, 60mmol) in water (15 ml) and methanol (120 ml). The solution was stirredat room temperature for 1 hr and then evaporated to dryness undervacuum. The residue was treated with 1 M HCl (30 ml) and extracted withdiethylether (3 portions of 100 ml). The combined extracts were washedwith 200 mM pH 7 phosphate buffer (50 ml), dried over magnesium sulfate,filtered and evaporated to give tricyclic amine-phenol 208 as a brownsolid, used crude in subsequent steps.

[0356] 8.2 Svnthesis of Tetrafluoro Ketone 210

[0357] A solution of aminophenol 208 (0.67 g, 2.9 mmol) andtetrafluorophthalic anhydride (0.64 g, 2.9 mmol) was refluxed in toluene(10 ml) for 3 hr. The solution was cooled to rt and the precipitatecollected, yielding tetrafluoro ketone 210 (0.92 g, 71%).

[0358] 8.3 Synthesis of Tetrafluoro 212

[0359] Phosphorous oxytrichloride (0.56 ml, 6 mmol) was added to asolution of F4 RAZ ketone 210 (0.91 g, 2 mmol) in chloroform (20 ml).The solution was stirred for 15 min., aminophenol 208 (0.46 g, 2 mmol)was added and the mixture was refluxed for 3 hr. The solution was cooledto rt and the reaction quenched with water (0.5 ml). Tetrafluoro dye 212was purified by normal phase chromatography (DCMM/eOH, 20:1) and furtherpurified by C18 reverse phase chromatography (NeOH/0.1 M TEAA, 9:1) toafford 212 as a metallic green solid (0.39 g, 30%, Abs. max 630 nm, Em.max 655 nm, 8M urea).

[0360] 8.4 Synthesis of Rhodamine Dye 232

[0361] Rhodamine dye 232 was synthesized from tetrafluoro dye 212 with4-(dimethylamino)pyridine and 4-carboxythiobenzene, as descfibed inSection 7.2, supra, and purified by C-8 reverse phase HPLC, eluting withan acetonitrile/0.1 M TEAA gradient (Em. max 665 nm, H₂O).

9. EXAMPLE Synthesis of Rhodamine Dye 234

[0362]

[0363] Rhodamline dye 234 was synthesized as illustrated in Schemes (I)and (II) from the appropriate aminophenol starting materials.

[0364] 9.1 Synthesis of Pyrrolodinyl Phenol 214

[0365] A solution of m-aminophenol (12.6 g, 115 mmol) was heated in1,4-dibromobutane (50 g, 230 mmol) at 130° C. for 18 hr. The mixtureswas cooled to room temperature and thie gum was triturated with etherand then ethyl acetate. The gum was dissolved in 120 ml of 1 M NAOH andextracted with ethyl acetate (100 ml). The layers were separated and theorganic phase was washed with water twice and then brine. After dryingover magnesium sulfate, the crude product was purified by silica gelchromatography (DCN/methanol 100:1) to give a pale yellow solid. Thesolid was refluxed in toluene (500 ml) and triethylamine (16 ml, 115mmol) for 2 hr., cooled to room temperature, and washed with water. Thesolvent was evaporated to leave pyrrolidinyl phenol 214 as a white solid(11 g, 60%). ¹H NMR (214, d6-DMSO) δ 8.98, 1H, s; 6.90, 1H, t; 5.95, 3H,m; 3.18, 4H, m; 1.95, 4H, m.

[0366] 9.2 Synthesis of Pyrrolidinyl Ketone 216

[0367] A solution of aminophenol 214 (0.74 g, 4.6 mmol) andtetrafluorophthalic anhydride (1 g, 4.6 mmol) was refluxed in toluene (5ml) for 3 hr. The solution was cooled to rt and the precipitatedcollected to yield pyrolidinyl ketone 216 (1.3 g, 77%).

[0368] 9.3 Svnthesis of Tetrafluoro 218

[0369] Phosphorous oxytrichloride (1 ml, 10 mmol) was added to asolution of pyrrolidinyl ketone 216 (1.3 g, 3.5 mmol) in chloroform (10ml). The solution was stirred for 15 min., aminophenol 214 (0.56 g. 3.5mmol) was added and the solution was refluxed for 4 hr. The solution wascooled to rt and the reaction quenched with water (0.5 ml). Tetrafluoro218 was purified by C18 reverse phase chromatography (MeOH/0.1 M TEAA,4: 1) to afford metallic green solid (0.73 g, 41%, Abs.max 576 nm, Em.max 594 nm, CH₃OH).

[0370] 9.4 Synthesis of Rhodamine Dye 234

[0371] Rhodamine dye 234 (Abs. max 590 nm, Em. max 606 nm, CH₃OH; Em.max 611 nm, H₂O) was synthesized from 218 as described in Section 7.2,supra, and purified by C-8 reverse phase HPLC, eluting with anacetonitrile/0.1 M TEAA gradient.

10. EXAMPLE Synthesis of Rhodamine Dye 236

[0372]

[0373] Rhodamine dye 236 was synthesized from 3-(bisbenzylamino)phenoland tetrafluorophthalic anhydride 114 as illustrated in Schemes I andII.

[0374] 10.1 Synthesis of Bis-Benzyl Tetrafluoro Ketone 222

[0375] A solution of 3-(bisbenzylamino)phenol (14.5 g, 50 mmol) andtetrafluorophthalic anhydride (11 g, 50 mmol) were refluxed in toluene(50 ml) for 18 hr. The solvent was evaporated and the residue purifiedby C18 reverse phase chromatography (MeOH/water, 7:3) to affordbis-benzyl tetrafluoro ketone 222 as a white solid (6.5 g, 25%).

[0376] 10.2 Synthesis of Tetra-benzyl Tetrafluoro 224

[0377] Phosphorous oxytrichloride (3.3 ml, 35 mmol) was added to asolution of bis-benzyl ketone 222 (6.0 g, 12 mmol) in chloroform (60ml). The solution was stirred for 15 min., 3-(bisbenzylamino)phenol (3.4g, 12 mmol) was added and the mixture was refluxed for 3 hr. Thesolution was cooled to room temperature and the reaction was quenchedwith water (1 ml). The solvent was evaporated and the residue purifiedby C18 reverse phase chromatography (NeOH/0.1 M TEAA, 9:1) to affordtetra-benzyl tetrafluoro dye 224 as a dark red solid (0.62 g, 7%, Abs.max 566 nm).

[0378] 10.3 Synthesis of Tetrafluoro 226

[0379] A suspension of dye 224 (0.62 g, 0.8 mmol) was heated in conc.HBr (20 ml) to 110° C. for 45 min. The reaction mixture was poured intoice-water and the precipitated was collected and purifed by C18 reversephase chromatography (CH₃OH: 0.1M TEAA, 3:2) to afford debenzylated dye226 as a metallic green solid (100 mg, 31%, Abs. max 521 nm).

[0380] 10.4 Synthesis of Rhodamine Dye 236

[0381] Rhodamine dye 236 was synthesized from 226 as described inSection 7.2, supra. Purification of 236 was accomplished on C18 silicagel with stepwise elution with 20-70% methanol vs. 0.1 Mtriethylammonium acetate. The dye 236 eluted between 30% and 50%ethanol. The solvent was evaporated and the residual blue oil wasprecipitated with ether to provide the title dye as a blue solid (85 mg,Em. max 545 nm, H₂O).

11. EXAMPLE Synthesis of Fret Dye 230

[0382]

[0383] 11.1 Synthesis of Succinimidyl (NS) Ester of 196

[0384] To a solution of dye 196 (5 mg) in DMF (100 μl) was addedsuccinmdyl tetramethyluronium tetrafluoroborate (20 mg) anddiisopropylamine (10 μL). TLC analysis on C2-reverse phase silica geleluting with 1:1 methanol: 0.1 M triethylamnmonium acetate coulddistinguish dye 196 (Rf=0.2) from 196 succinimidyl ester (Rf=0). After 1hr, the reaction appeared to be complete and was partitioned between 5%HCl and dichloromethane. The organic layer was dried over Na2SO₄ and thesolvent evaporated to yield the 196 NHS ester as a blue solid.

[0385] 11.2 Synthesis of FRET Dye 230

[0386] Energy-transfer (FRET) dye 230 is prepared by coupling 196 NHSester and 4-aminomethylbenzoic acid and4′-aminomethyl-6-carboxyfluorescein (Molecular Probes Inc., Eugene,Oreg.), according to methods described in U.S. Pat. No. 5,863,727. Forexample, 1 μmole of 196 NHS dissolved in 250 μl of DMSO is added to asolution of 2 μmole 4′-aminomethyl-6-carboxyfluorescein in 100 μl DMSOand 20 μl triethylamine. After mixing the solution is let stand forabout 12 hours, monitoring the progress of coupling by reverse phaseHBLC.

12. EXAMPLE Synthesis of Fret Dye 230-ddATP

[0387]

[0388] FRET dye 230 is activated as the N-HS ester by the method ofExample supra 11.1 and coupled to 7-deaza-7-aminopropargyl, 2′-3′dideoxyadenosine-5′-triphosphate according to the methods in U.S. Pat.No. 5,821,356 and 5,770,716 to give FRET dye 230-ddATP. Alternatively,4′-trifluoroacetamidomethyl 6-carboxyfluorescein NHS ester is coupledwith 7-deaza-7-aminopropargyl, 2′-3′ dideoxyadenosine-5′-triphosphate togive the intermediate dye-nucleotide. The trifluoroacetyl protectinggroup is removed under basic conditions and the intermediate dyenucleotide is coupled with 230 NHS ester to give FRET dye 230-ddATP by atwo-step method of conjugating the energy-transfer dye to a nucleotide.

13. EXAMPLE Sanger-Type Sequencing Using an Energy Transfer Dye

[0389] Following the methods described in U.S. Pat. Nos. 5,821,356 and5,366,860, Sanger-type terminator sequencing was performed on pGEM (PEBiosystems, Foster City, Calif.) using Taq FS polymerase (PE Biosystems,Foster City, Calif.), a mixture of four dNTPs, an unlabeled sequencingprimer and a single labeled terminator (6-FAM-230-ddATP). A plot of theresultant sequencing data, obtained on an ABI PRISM Model 310 instrument(PE Biosystems, Foster City, Calif.) is provided in FIG. 1.

14. EXAMPLE Four-Color Sanger-Type Sequencing

[0390] Following the methods described in U.S. Pat. Nos. 5,821,356 and5,366,860, four-color Sanger-type terminator sequencing was performed onpGEMI (PE Biosystems, Foster City, Calif.) using Taq FS polymerase (PEBiosystems, Foster City, Calif.), a mixture of four dNTPs, an unlabeledsequencing primer and a mixture of four, 3′-fluoro,spectrally-resolvable labeled terminators (6-FAM-230-ddATP;5-FAM-236-7-deaza-ddGTP; 6-FAM-JON-ddTTP; 6-FAM-ROX-ddCTP). A plot ofthe resultant sequencing data, obtained on an ABI PRISM Model 310instrument (PE Biosystems, Foster City, Calif.) is provided in FIG. 2.

[0391] As can be seen in FIG. 2, all of the dye-labeled polynucleotidesexhibit significant fluorescence intensity. Moreover, the differentdye-labeled polynucleotide exhibit sufficiently similar mobilities,resulting in good resolution.

15. EXAMPLE Anti-Human Antibody-Dye Conjugate Detection

[0392] Anti-Human IL-8 Antibody-Dye—196 Conjugate

[0393] Polyclonal anti-human IL-8 antibody (R&D Systems, Minneapolis,Minn.; 0.5 mg in 0.5 ml PBS) was incubated with 50 μl of 1 M Na₂CO₃ and105 μg of dye 196 for 1 hour at room temperature, in the dark. Theantibody-dye—196 conjugate (Abs. max 666 nm, H₂O) was separated from thefree dye using a sephadex G-50 (fine) size exclusion column. Theconjugate was passed through a 0.2 μL filter and stored at 4° C. in PBS.The concentration of the antibody was determined according to thefollowing equation: [A₂₈₀-(0.82×A640)]/170,000, where 0.82=A₂₈₀/A₆₄₀ ofthe free dye and 170,000 is the extinction coefficient for the antibody.The concentration of the dye was determined according to the followingequation: A₆₄₀/120,000, where 120,000 is the extinction coefficient fordye 196. The dye 196-labeled polyclonal anti-human IL-8 antibodyconcentration was 0.18 mg/ml with an F/P ratio of 2.3/1.

[0394] Preparation of Monoclonal Anti-Human IL-8 Antibody Coated Beads

[0395] Goat anti-mouse IgG (Fc) polystyrene beads (200 ml of 0.5% w/v; 6μm bead diameter; Spherotech) were first washed 3 times bycentrifugation and resuspension of the bead pellet with 1 ml PBS. 4 μgof monoclonal anti-human IL-8 antibodies %&D Systems) was added to thebead suspension for a final volume of 1 ml. After incubation for 16 hrsat room temperature with gentle mixing, the monoclonal antibody-coatedbeads were washed 2 times as described above and resuspended in 1 mlPBS. The final bead concentration was 0.1% w/v or 8×10⁶ beads/ml. Themonoclonal antibody-coated beads were stable for approximately 1 monthwhen stored at 4° C.

[0396] Fluorescent-Linked Immunosorbent Assay (FLISA)

[0397] To generate a standard curve, a two-fold serial dilution of humanIL-8 peptide (R&D Systems) in 50 μL FLISA buffer (PBS containing 1 mg/mlBSA, 0.35 M NaCl, 0.1% Tween 20, and 0.01% NaN₃) were aliquoted into a96-well plate (Corning Costar No. 3904). Subsequently, 50 μl of amixture containing monoclonal anti-human IL-8 antibody-coated beads anddye 196-labeled polyclonal anti-human IL-8 antibodies in FLISA bufferwas then added to each well. The final concentrations in a total volumeof 100 μl per well were 64,000 antibody-coated beads/ml and 0.11 μg/mlof dye 196-labeled polyclonal anti-human IL-8 antibodies. The serialdilution for IL-8 peptide ranged from 2,000-2.0 pg/ml. The assaycontained eight replicates, and control wells containing no peptide wereincluded. After incubating overnight at room temperature in the dark,the 96-well plate was scanned using the FMAT 8100 HTS System (PEBiosystems, Foster City, Calif.), a macroconfocal imaging systemequipped with a 633-nm HeNe laser and 2-channel fluorescence detection(FL1: 650-685 nm, FL2: 685-720 nm). A 1 mm² area of each well is rasterscanned at a depth of focus of 100 μm and at a rate of 1 sec/well, with256 scan lines generated. The fluorescence intensity associated witheach bead is then obtained from the digitized images and the averagefluorescence intensity per bead is calculated per well. A log vs loggraph of the average fluorescence intensity in FL1 vs pg/ml of the IL-8peptide is shown in FIG. 4. The linear dynamic range of the assay is250-3.9 pg/ml IL-8.

[0398] While the present invention has been described by reference tothe preferred embodiments and examples detailed above, it is to beunderstood that these examples are intended in an illustrative, ratherthan limiting, sense. It is contemplated that modifications that do notdepart from the spirit of the invention will readily occur to those ofskill in the art. Any such modifications are intended to fall within thescope of the appended claims.

[0399] All of the references, publications and patents cited in thespecification are incorporated herein by reference to the same extent asif each reference were individually incorporated by reference.

We claim:
 1. A rhodamine dye or a salt thereof, comprising arhodamine-type parent xanthene ring having attached to the xanthene C9carbon a phenyl group that is further substituted with an ortho carboxyor ortho sulfonate group or a salt thereof, one to three substituted orunsubstituted aminopyridimnum groups and a substituted or unsubstitutedalkylthio, arylthio or heteroarylthio group, said rhodamine dyeoptionally including one or more linking moieties.
 2. The rhodamine dyeof claim 1 which comprises the structure:

wherein: n is 1, 2, or 3; Y is a rhodamine-type parent xanthene ringattached to the illustrated phenyl group at the xanthene C9 carbon; eachR is independently selected from the group consisting of (C₁-C₆) alkyland heteroalkl, (C₁-C₂₀) aryl and heteroaryl (C₆-C₂₆) arylalkyl andheteroalkyl, (C₅-C₂₀) arylaryl and heteroaryl-heteroaryl, or when takentogether, R is (C₄-C₁₀) alkyldiyl, (C₄-C₁₀) alkyleno, heteroalkyldiyland heteroalkyleno; S is sulfuir; Z is (C₁-C₁₂) alkyl, (C₁-C₁₂) alkylsubstituted with one or more of the same or different W¹ groups,(C₅-C₂₀) aryl and heteroaryl, and (C₅-C₂₀) aryl and heteroarylsubstituted with one or more of the same or different W² groups; W¹ isselected from the group consisting of —X, —R, ═O, —OR, —SR, ═S, —NRR,═NR, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻,—C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR; W² isselected from the group consisting of —R, —OR, —SR, —NRR, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)OF,—C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR; each X isindependently a halogen; and Y or Z is optionally substituted with Lwhere L is a bond or a linker.
 3. The rhodamine dye of claim 2 in whichL is selected from a hydrophobic moiety, a charged group, a member of apair of specific binding molecules, a photo-activatable group and areactive functional group.
 4. The rhodamine dye of claim 2 where Z hasthe form Z¹—L—R_(X), or a salt thereof, wherein: Z¹ is (C₁-C₁₂)alkyldiyl, (C₁-C₁₂) alkyldiyl independently substituted with one or moreof the same or different W¹ groups, or (C₅-C₁₄) aryldiyl, and aryldiyl,heteroaryldiyl and heteroaryldiyl independently substituted with one ormore of the same or different W² groups; L is a bond or a linker; andR_(X) is a reactive functional group.
 5. The rhodamine dye of claim 4 inwhich Y is selected from:

and a salt thereof, wherein: R¹and le When taken alone, areindependently hydrogen or (C₁-C₆) alkyl; R³and R^(3′) when taken alone,are independently selected from the group consisting of hydrogen,(C₁-C₆) alkyl, (C₁-C₁₄) aryl and arylaryl, or when taken together is(C₄-C₆) alkyidyl or alkyleno, or when individually taken together withR² or R⁴ is (C₂-C₆) alkyldiyl or (C₂-C₆) alkyleno; R⁴, when taken alone,is selected from the group consisting of hydrogen and (C₁-C₆) alkyl, orwhen taken together with R³ or R^(3′) is (C₂-C₆) alkyldiyl or alkyleno;R⁵, when taken alone, is selected from the group consisting of hydrogenand (C₁-C₆) alkyl, or when taken together with R⁶ or R^(6′) is (C₂-C₆)alkyldiyl or alkyleno; R⁶ and R^(6′) when taken alone, are selected fromthe group consisting of hydrogen, (C₁-C₆) alkyl, (C₅-C₁₄) aryl andarylaryl, or when taken together are (C₄-C₆) alkyldiyl or alkyleno, orwhen individually taken together with R⁵ or R⁷ is (C₂-C₆) alkyldiyl oralkyleno; R⁷, when taken alone, is selected from the group consisting ofhydrogen and (C₁-C₆) alkyl, or when taken together with le or R⁶ is(C₂-C₆) alkyldiyl or alkyleno; R⁸, when taken alone, is selected fromthe group consisting of hydrogen and (C₁-C₆) alkyl; R⁹ indicates thepoint of attachment to the ortho-carboxyphenyl bottom ring; and R¹⁰,R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are eachindependently selected from the group consisting of hydrogen and (C₁-C₆)alkyl, or when R¹⁰, R¹¹, R¹² and R¹³ taken together are (C₅-C₁₄) arylenoor (C₅-C₁₄) aryleno substituted with one or more of the same ordifferent (C₁-C₆) alkyl, or when R¹⁸, R¹⁹, R²⁰ and R²¹ taken togetherare (C₅-C₁₄) aryleno or aryleno substituted with one or more of the sameor different (C₁-C₆) alkyl.
 6. The rhodamine dye of claim 5 wherein R²,when taken together with R³ or R^(3′) is (C₂-C₆) alkyldiyl or alkyleno.7. The rhodamine dye of claim 6 wherein: alkyl is methanyl, ethanyl orpropanyl; aryl is phenyl or naphthyl; arylaryl is biphenyl; alkyldiyl oralkyleno bridges formed by taking R² together with R³ or R^(3′), R⁷together with R⁶ or R^(6′), or R⁴ together with and R³ or R^(3′), areethano, propano, 1,1-dimethylethano, 1,1-dimethylpropano and1,1,3-trimethylpropano; aryleno bridges formed by taking R¹ togetherwith R² are benzo or naphtho; alkyldiyl or alkyleno bridge formed bytaking R³ together with R^(3′), or R⁶ together with R⁶, is butano;alkyldiyl or alkyleno bridges formed by taking R⁵ together with R⁶ orR^(6′) are ethano, propano, 1,1-dimethylethano, 1,1-dimethylpropano and1,1,3-trimethylpropano; and aryleno bridge formed by taking R¹⁰, R¹¹,R¹² and R¹³ together, or R¹⁸, R¹⁹, R²⁰ and R²¹ together, is benzo. 8.The rhodamine dye of claim 6 in which L is a bond.
 9. The rhodamine dyeof claim 4 in which R_(X) is selected from the group consisting ofcarboxyl, carboxylate, ester and activated ester.
 10. The rhodamine dyeof claim 4 in which Z¹ is selected from the group consisting of (C₁-C₁₂)alkyleno, (C₁-C₁₂) alkano, (C₅-C₁₀) aryldiyl and heteroaryldiyl,phenyldiyl, phena-1,4-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyland purindiyl.
 11. The rhodamine dye of claim 4 in which Y is selectedfrom the group consisting of:


12. The rhodamine dye of claim 4 in which L is a bond.
 13. The rhodaminedye of claim 4 in which R_(X) is selected from the group consisting ofcarboxyl, carboxylate, ester and activated ester.
 14. The rhodamine dyeof claim 4 in which Z¹ is selected from the group consisting of (C₁-C₁₂)alkyleno, (C₁-C₁₂) alkano, (C₅-C₁₀) aryldiyl and heteroarylidyl,phenylidyl, phena-14-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyland purindiyl.
 15. The rhodamine dye of claim 4 which comprises thestructure:

or a salt thereof.
 16. The rhodamine dye of claim 15 in which Y isselected from the group consisting of Y-1, Y-2, Y-3 and Y-4.
 17. Therhodamine dye of claim 15 in which Y is selected from the groupconsisting of Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a,Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a. 18.The rhodamine dye of claim 2 which has the structure:

wherein: Y¹ is a rhodamine-type parent xanthene ring attach ed to theillustrated phenyl group at the xanthene C9 carbon; L is a bond orlinker attached to a xanthene nitrogen atom or a xanthene C4 carbon; nis 1, 2, or 3; and R_(X) is a reactive functional group.
 19. Therhodamine dye of claim 18 in which Y¹ is selected from the groupconsisting of:

wherein the dashed line at the nitrogen or C4 atom indicates the pointof attachment of substituent L.
 20. The rhodamine dye of claim 19wherein: alkyl is methanyl, ethanyl or propanyl; aryl is phenyl ornaphthyl; arylaryl is biphenyl; alkyldiyl or alkyleno bridges formed bytaking R² together with R³, R⁴ together with R^(3′), R⁵ together withR⁶, or R⁷ together with R^(6′), are ethano, propano, 1,1-dimethylethano,1,1-dimethylpropano and 1,1,3-trimethylpropano; aryleno bridges formedby taking R¹⁰, R¹¹, R¹² and R¹³ together or R¹⁸, R¹⁹, R²⁰ and R²¹together are benzo.
 21. The rhodamine dye of claim 18 in which L isselected from the group consisting of (C₁-C₆) alkyldiyl, (C₁-C₆) alkano,(C₅-C₂₀) aryldiyl, phenyldiyl, phena-1,4-diyl, naphthyldiyl,naphtha-2,6-diyl, naphtha-2,7-diyl, (C₆-C₂₆) arylalkyldiyl —(CH)_(i)-Φ-and —(CH)_(i)-ψ-, where each i is independently an integer from 1 to 6,φ is (C₅-C₂₀) aryldiyl, phenyldiyl or phena-1,4-diyl and ψ isnaphthyldiyl, naphtha-2,6-diyl or naphtha-2,7-diyl.
 22. The rhodaminedye of claim 18 in which R_(X) is selected from the group consisting ofcarboxyl, carboxylate, ester and activated ester.
 23. The rhodamine dyeof claim 18 in which Z is selected from the group consisting of (C₁-C₁₂)alkyl, (C₁-C₁₂) alkanyl, (C₅-C₁₀) aryl and heteroaryl, phenyl, naphthyl,naphth-1-yl, naphth-2-yl, pyridyl and purinyl.
 24. The rhodamine dye ofclaim 18 in which Y¹ is selected from the group consisting of:

wherein R⁹ and the dash at the nitrogen or C4 atom indicates the pointof attachment of L.
 25. The rhodamine dye of claim 18 which has thestructure:


26. The rhodamine dye of claim 25 in which Y¹ is selected from the groupconsisting of Y-1b, Y-2b, Y-3b, Y-4b, Y-1c, Y-2c, Y-3c and Y-4c.
 27. Therhodamine dye of claim 25 in which Y¹ is selected from the groupconsisting of Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c,Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c,Y-36b, Y-36c, Y-3b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b andY-46c.
 28. An energy-transfer dye pair comprising a donor dye linked toan acceptor dye, wherein the donor dye or the acceptor dye is a compoundaccording to claim 1 and either or both of said donor and acceptor dyesinclude an optional linking moiety.
 29. The dye pair of claim 28 whichhas the structure:

or a salt thereof, wherein: R⁴¹ is a covalent linkage formed uponreaction between a nucleophile and an electrophile; L″ is a bond or alinker; n is 1,2, or 3; and DD/AD is a donor dye or an acceptor dyewhich includes a linking moiety.
 30. The dye pair of claim 29 in which Yis selected from the group consisting of Y-1, Y-2, Y-3, Y-4, Y-20a,Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31 a, Y-34a, Y-35a, Y-36a, Y-39a,Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.
 31. The dye pair of claim29 in which L is a bond.
 32. The dye pair of claim 29 in which R⁴¹ hasthe formula —C(O)NR⁴⁵—, where R⁴⁵ is hydrogen or (C₁-C₆) alkyl.
 33. Thedye pair of claim 29 in which Z¹ is selected from the group consistingof (C₁-C₁₂) alkyleno, (C₁-C₁₂) alkano, (C₅-C₁₀) aryldiyl andheteroaryldiyl, phenyldiyl, phena-1,4-diyl, naphthadiyl,naphtha-2,6-diyl, pyridindiyl and purindiyl.
 34. The dye pair of claim29 in which L″ is —R⁴³—Z³—C(O)—R⁴⁴—R⁴⁵—, wherein R⁴³ is (C₁-C₆)alkyldiyl, preferably (C₁-C₃) alkano, and is bonded to R⁴², where R⁴² isO, S or NH; Z³ is 5-6 membered cyclic alkenyldiyl and heteroalkenyldiyl,(C₁-C14) aryldiyl and heteroaryldiyl; R⁴⁴ is O, S or NH; and R⁴⁵ is(C₁-C₆) alkyldiyl, preferably (C₁-C₃) alkano.
 35. The dye pair of claim29 in which DD/AD is a fluorescein dye in which the linking moiety is areactive functional group and wherein L″ is attached to the fluoresceindye at the xanthene C4 carbon.
 36. The dye pair of claim 29 which hasthe structure:

wherein, R⁵⁰ is a carboxyl, a salt, ester or activated ester thereof.37. The dye pair of claim 36 in which Y is selected from the groupconsisting of Y-1, Y-2, Y-3, Y-4, Y-20a, Y-21a, Y-22a, Y-23a, Y-24a,Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41 a, Y-42a, Y-43a, Y-44a,Y-45a and Y-46a.
 38. The dye pair of claim 28 which has the structure:

wherein: R⁴¹ is a covalent linkage formed upon reaction between anucleophile and an electrophile; L″ is a bond or a linker; n is 1, 2, or3; and DD/AD is a donor dye or an acceptor dye which includes a linkingmoiety.
 39. The dye pair of claim 38 in which Y¹ is selected from thegroup consisting of Y-1b, Y-2b, Y-3b, Y-4b, Y-1c, Y-2c, Y-3c, Y-4c,Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c,Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c,Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c. 40.The dye pair of claim 38 in which L is (C₁-C₆) alkyldiyl or (C₁-C₃)alkano.
 41. The dye pair of claim 38 in which R⁴¹ is an amide of theformula —C(O)NR⁴⁵, where R⁴⁵ is hydrogen or (C₁-C₆) alkyl.
 42. The dyepair of claim 38 in which Z is selected from the group consisting of(C₁-C₁₂) alkyl, (C₁-C₁₂) alkanyl, (C₅-C₁₀) aryl and heteroaryl, phenyl,naphthyl, naphth-1yl, naphth-2-yl, pyridyl and purinyl.
 43. The dye pairof claim 38 in which L″ is —R⁴³—Z³—C(O)—R⁴⁴—R⁴⁵—, wherein R⁴³ is (C₁-C₆)alkydiyl, preferably (C₁-C₃) alkano, and is bonded to R⁴², where R⁴² isO, S or NH; Z³ is 5-6 membered cyclic alkenyldiyl and heteroalkenyldiyl,(C₅-C₁₄) aryldiyl and heteroaryldiyl; R⁴⁴ is O, S or NH; and R⁴⁵ is(C₁-C₆) alkyldiyl, preferably (C₁-C₃) alkano.
 44. The dye pair of claim38 in which DD/AD is a fluorescein dye in which the linking moiety is areactive group R_(X) and wherein L″ is attached to the fluorescein dyeat the xanthene C5 carbon.
 45. The dye pair of claim 38 which has thestructure:

wherein: Y¹ is selected from the group consisting of Y-20b, Y-20c,Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c,Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b,Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c; and R⁵⁰ is acarboxyl, a salt, ester or activated ester thereof.
 46. The dye pair ofclaim 45 in which Y¹ is selected from the group consisting of Y-1b,Y-2b, Y-3b, Y-4b, Y-1c, Y-2c, Y-3c, Y-4c, Y-20b, Y-20c, Y-21b, Y-21c,Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c,Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c,Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.
 47. A labeled nucleoside/tide ornucleoside/tide analog comprising the rhodamine dye of claim 2 where Zhas the form Z¹—L—R⁴⁶—L′—NUC, wherein: R⁴⁶ is a linkage formed byreaction between an electrophile and a nucleophile; and —L′—NUC takentogether has the structure:

wherein: B is a nucleobase; L′ is (C₁-C₂₀) alkyldiyl andheteroalkyldiyl, (C₁-C₂₀) alkyleno and heteroalkyleno, (C₂-C₂₀) alkynoand heteroalkyno, or (C₂-C₂₀) alkeno and heteroalkeno; R₇₀ and R₇₁, whentaken alone, are each independently selected from the group consistingof hydrogen, hydroxyl and a moiety which blocks polymerase-mediatedtemplate-directed polymerization, or when taken together form a bondsuch that the illustrated sugar is 2′,3′-didehydroribose; and R₇₂ isselected from the group consisting of hydroxyl, a phosphate ester havingthe formula

where a is an integer from 0 to 2, and a phosphate ester analog, or asalt thereof.
 48. The labeled nucleoside/tide or nucleoside/tide analogof claim 47 where Z has the form Z¹—L—R⁴¹—L″—DD/AD—L³—R⁴⁶—L′—NUC, or asalt thereof, wherein: R⁴¹ is a covalent linkage formed upon reactionbetween a nucleophile and an electrophile; L″ is a bond or a linker;DD/AD is a donor dye or an acceptor dye which includes a linking moiety;and. L³ is a bond or a linker.
 49. The labeled nucleoside/tide ornucleoside/tide analog of claim 47 where Y has the formY¹—R⁴¹—L″—DD/AD—L³—R⁴⁶—L′—NUC, or a salt thereof wherein: Y¹ is Y-1,Y-2, Y-3, or Y-4; R⁴¹ is a covalent linkage formed upon reaction betweena nucleophile and an electrophile; L″ is a bond or a linker; DD/AD is adonor dye or an acceptor dye which includes a linking moiety; and. L³ isa bond or a linker.
 50. A labeled nucleoside/tide or nucleoside/tideanalog of claim 47 where Y has the form Y¹—R⁴¹—L″13 DD/AD and Z has theform Z¹—L—R⁴⁶—L′—NUC, or a salt thereof; wherein: Y¹ is Y-1, Y-2, Y-3,or Y-4; R⁴¹ is a covalent linkage formed upon reaction between anucleophile and an electrophile; L″ is a bond or a linker; DD/AD is adonor dye or an acceptor dye which includes a linking moiety; and Z¹ is(C₁-C₁₂) alkyldiyl, (C₁-C₁₂) alkyldiyl independently substituted withone or more of the same or different W¹ groups, (C₅-C₁₄) aryldiyl, and(C₅-C₁₄) aryldiyl, heteroaryldiyl and heteroaryldiyl independentlysubstituted with one or more of the same or different W² groups.
 51. Thelabeled nucleoside/tide or nucleoside/tide analog of claim 47, 48, 49 or50 which is enzymatically incorporatable.
 52. The labelednucleoside/tide or nucleoside/tide analog of claim 47, 48, 49 or 50which is a terminator.
 53. The lableled nucleoside/tide ornucleoside/tide analog of claim 47, 48, 49 or 50 which is enzymaticallyextendable.
 54. The labeled nucleoside/tide or nucleoside/tide analog ofclaim 47 in which L′ is selected from the group consisting of:propargyl, where the terminal sp carbon is covalently attached tonucleobase B and the terminal methylene (sp³) carbon is covalentlyattached to F_(x); and —C≡C—CH₂—O—CH₂—CH₂—NR⁴⁷—R⁴⁸—, where R⁴⁷ ishydrogen or (C₁-C₆) alkyl and R⁴⁸ is —C(O)—(CH₂)_(r)—, —C(O)—CHR⁴⁹—,—C(O)—C≡C—CH₂— or —C(O)-φ-(CH₂)_(r)—, where each r is independently aninteger from 1 to 5 and φ is C₆ aryldiyl orheteroaryldiyl and R⁴⁹ ishydrogen, (C₁-C₆) alkyl or a side chain of an encoding or non-encodingamino acid, and where the terminal sp carbon is covalently attached tonucleobase B and the other terminal group is covalently attached toF_(x).
 55. The labeled nucleoside/tide or nucleoside/tide analog ofclaim 48 or claim 49 in which L³ is a bond, R⁴⁶ the formula —C(O)—NHR⁵¹,where R⁵¹ is hydrogen or (C₁-C₆) alkyl.
 56. The labeled nucleoside/tideor nucleoside/tide analog of claim 47 in which nucleobase B is a purine,a 7-deazapurine, an 8-aza, 7-deazapurine, a pyrimidine, a normalnucleobase or a common analog of a normal nucleobase.
 57. The labelednucleoside/tide or nucleoside/tide analog of claim 47 or claim 48 inwhich Y is selected from the group consisting of Y-1, Y-2, Y-3 and Y-4.58. The labeled nucleoside/tide or nucleoside/tide analog of claim 47 orclaim 48 in which Y is selected from the group consisting of Y-20a,Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a,Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.
 59. The labelednucleoside/tide or nucleoside/tide analog of claim 49 or claim 50 inwhich Y¹ is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y4b,Y-1c, Y-2c, Y-3c and Y-4c.
 60. The labeled nucleoside/tide ornucleoside/tide analog of claim 49 or claim 50 in which Y¹ is selectedfrom the group consisting of Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c,Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c,Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b,Y-46b and Y-46c.
 61. A polynucleotide labeled with a rhodamine dyeaccording to claim 1 or an energy-transfer dye pair according to claim28.
 62. A method of generating a labeled primer extension product,comprising the step of enzymatically extending a primer-target hybrid inthe presence of a mixture of enzymatically-extendable nucleotidescapable of supporting continuous primer extension and a terminator,wherein said primer or said terminator is labeled with a rhodamine dyeaccording to claim 1 or an energy-transfer dye pair according to claim28.
 63. The method of claim 62 in which the terminator has thestructure:

wherein R₇₀ and R₇₁, when taken alone, are each independently selectedfrom the group consisting of hydrogen, halide, and any moiety whichblocks polymerase-mediated template-directed polymerization.
 64. Themethod of claim 62 in which the terminator is a mixture of fourdifferent terminators, one which terminates at a template A, one whichterminates at a template G, one which terminates at a template C and onewhich terminates at a template T or U.
 65. The method of claim 62 inwhich each of the four different terminators is labeled ith a different,spectrally-resolvable fluorophore.
 66. A labelled rhodaminedye-polypeptide conjugate comprising the rhodamine dye of claim 1 and apolypeptide, wherein the polypeptide is selected from the groupconsisting of a peptide, a protein, and an antibody.
 67. A method ofdetecting a rhodamine dye-antibody conjugate, in which said conjugate isa rhodamine dye-antibody conjugate according to claim 66, comprising thesteps of: (a) binding the conjugate to a peptide or protein, and (b)detecting the rhodamine dye-antibody conjugate bound to the peptide orprotein.
 68. The method of claim 67 in which the conjugate is bound tothe peptide or protein in the presence of a second antibody specific forbinding said peptide or protein.
 69. The method of claim 68 in which thesecond antibody is bound to a solid bead or particle.